Bacterial extract for respiratory disorders and process for its preparation

ABSTRACT

The present invention relates to an extract from bacterial strains, such as  Staphylococcus, Moraxella, Klebsiella, Streptococcus , and  Haemophilus . The extract is useful as a treatment for indications such as respiratory disorders, compositions comprising the extract, and processes of making the extract from media that do not pose a risk of prion diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/904,789, filed Mar. 5, 2007.

FIELD OF THE INVENTION

The present invention relates to extracts from bacterial strains usefulas a treatment for indications such as respiratory disorders,compositions comprising the extracts, and processes of making theextracts using media that do not pose a risk of prion diseases.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to compositions comprising bacterialextracts useful for treating medical conditions such as respiratorydisorders. The extracts may comprise bacterial lysates from cultureschosen from the following species:

-   -   Moraxella (Branhamella) catarrhalis, Moraxella (Moraxella)        catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,        Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus        pyogenes, Streptococcus sanguinis, Staphylococcus Hemolyticus,        Enterococcus faecalis, Streptococcus mutans, Streptococcus        anginosus, Streptococcus mitis, Streptococcus salivarius (aka.        Streptococcus viridans), Neisseria sicca, Hemophilus        parainfluenzae, Actinobacillus (Hemophilus)        actinomycetemcomitans, and Eikenella corrodens.

In some embodiments, the extracts comprise at least one strain from eachof the above species of bacteria, while in other embodiments, one ormore specific strains from the list above may be removed or substitutedwith one or more different strains. Some embodiments of the presentinvention comprise an extract obtained from each of the followingbacterial strains: Moraxella (Branhamella) catarrhalis 3622, Moraxella(Branhamella) catarrhalis 3625, Moraxella (Branhamella) catarrhalisI-045, Haemophilus influenzae 8467, Klebsiella pneumoniae ssp. ozaenae5050, Klebsiella pneumoniae 204, Klebsiella pneumoniae 5056,Staphylococcus aureus I-049, Staphylococcus aureus I-050, Staphylococcusaureus I-051, Staphylococcus aureus I-052, Staphylococcus aureus I-053,Staphylococcus aureus I-054, Streptococcus (Diplococcus) pneumoniae7465, Streptococcus (Diplococcus) pneumoniae 7466, Streptococcus(Diplococcus) pneumoniae 7978, Streptococcus (Diplococcus) pneumoniae10319, Streptococcus pyogenes 8191, Streptococcus sanguinis I-046,Streptococcus sanguinis I-047, Streptococcus sanguinis I-048,Staphylococcus Hemolyticus 11042, Enterococcus faecalis 103015,Streptococcus mutans 10449, Streptococcus anginosus 10713, Streptococcusmitis 12261, Streptococcus salivarius 102503, Neisseria sicca 103345,Haemophilus parainfluenzae 7857, Actinobacillus (Hemophilus)actinomycetemcomitans 52.105, and Eikenella corrodens 10596. Thosestrains are deposited according to the Budapest Treaty. The strainsindicated in the list with I-number were indexed by the CollectionNationale de Culture des Microorganismes at the Institut Pasteur, 25 ruedu Dr. Roux, 75724 Paris, France. All of the other strains were indexedby the National Collection of Type Cultures in London.

In some embodiments, one or more of the specific strains listed abovemay be omitted, or substituted with a different strain from the samespecies or from a different species of bacteria. For example, in someembodiments, one or more or even all of strains StaphylococcusHemolyticus 11042, Enterococcus faecalis 103015, Streptococcus mutans10449, Streptococcus anginosus 10713, Streptococcus mitis 12261,Streptococcus salivarius 102503, Neisseria sicca 103345, Haemophilusparainfluenzae 7857, Actinobacillus (Hemophilus) actinomycetemcomitans52.105, and Eikenella corrodens 10596 may be omitted. In otherembodiments, one or more Moraxella, Klebsiella, Staphylococcus aureus,Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcussanguinis strains may be omitted. Further, to aid digestion, aLactobacillus strain or another strain of bacteria may also be used.

The extracts may be obtained by a process of alkaline lysis after cellsare grown to a suitable optical density in a culture medium. In someembodiments, the bacteria are each grown on a medium that does not posea risk of prion-related diseases or a risk of other diseases that may betransmitted through ingesting products obtained from animal-based media.For example, in some embodiments a vegetable-based medium is used togrow the cells, such as a soya-based medium. In other embodiments, asynthetic medium is used for cell growth. In yet other embodiments, amedium may include biological extracts such as yeast extract and horseserum, which also do not pose such disease risks.

The lysates may also be filtered to remove nucleic acids and largercellular debris. In consequence of the filtration, in some embodiments,the amount of nucleic acid present in the extracts is less than 100μg/mL. In some embodiments, insolubilized compounds such as cell walldebris and insufficiently degraded lipopolysaccharide (LPS) are alsoremoved by the filtration. Hence, in some embodiments, the resultingextract comprises soluble molecular components and does not containsignificant amounts of insoluble or particulate material.

Saccharide components may be preserved in the extracts, includinglipopolysaccharide (LPS) components. During the lysis process,saccharides may become chemically modified, for example, cleaved intosmaller structures or substituted with other functional groups.

Racemization of amino acids during the lysis process also createsD-amino acids from the naturally occurring L-amino acids found innatural proteins. D-amino acids can be beneficial in increasingbioavailability of the extracts, as proteins constituted principally orpartially from D-amino acids are not efficiently digested in themammalian gut. Thus, antigenic molecules in the extracts that arechemically modified during lysis to contain D-amino acids remain in thepatient's body for a longer time, allowing potentially for a strongerimmunostimulating action.

While bacterial extracts have been used in the prior art to stimulatethe immune system against respiratory diseases, there has been a need tobetter standardize and control those extracts in order to make themsafer, more effective, and longer lasting. For instance, it waspreviously thought that saccharide components, including potentiallytoxic lipopolysaccharide (LPS) components should be removed frombacterial extracts for safety reasons. (See, e.g., U.S. Pat. No.5,424,287.) However, the instant invention provides a process thatresults in sufficient chemical modifications of LPS components thatsaccharides be safely retained. Retaining those components may improveefficacy and provide additional antigens to the extracts.

For example, the inventors have discovered that adjusting the pH and thetime of lysis may allow for sufficient degradation of potentiallyallergenic or toxic cell wall components. Prior lysis conditions atlower pH's or shorter times, in contrast, produced extracts in whichcell wall components and saccharides were insufficiently chemicallymodified. (See, e.g., GB 2 021 415 A.) The resulting extracts were tooallergenic to be safely administered to patients. In general, theinventors have discovered that products lysed at too low a pH and/or attoo short a time had higher toxicity, lower protein extraction, andlower filterability.

The filtration process may also influence the properties of theresulting extract, as the pore size of the filter, and in some cases,the chemical properties of the filter surface, may alter the type ofmaterials that were removed and retained. For example, some embodimentsof the instant invention use a filtration process designed to retainsaccharides but to remove other molecular components such as nucleicacids.

Thus, the instant invention provides parameters that standardize thebacterial extracts to help maintain consistent safety and efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A diagram of a tangential flow filtration (TFF) system forpreparation of bacterial extracts following lysis of bacteria. Thediagram shows two different configurations for filters: a parallel modewhere all filters work simultaneously and a serpentine mode wherefilters are configured in a serial mode.

FIGS. 2A-2B: IL-6 and TNF-α production by human PBMC incubated withserial dilutions of a purified mixture of extracts from Examples 2.2,3.6, 3.7, 3.8, 3.9, and 3.10.

FIG. 3: Survival of mice challenged with virus H1N1 during 3 weeks afterthe infection. Mc Nemar: test *p=0.023 for 10 mg treatment vs control.

FIG. 4: Effects of NaOH concentration, amount of biomass (expressed ingrams dry weight per milliliter), and duration of lysis (in hours) onthe biological activities of purified HAIN 8467 extracts (Examples 2.1,2.5, 2.8) in a nitric oxide bioassay in murine macrophages.

FIG. 5: Nitric oxide bioassay in murine macrophages of a purifiedmixture of Diplococcus pneumonia extracts (Example 3.6).

FIG. 6: Biological activities of the extracts of Example 3.1 and Example3.3 (labeled 3a and 3c, respectively) in a nitric oxide bioassay inmurine macrophages.

FIG. 7: Biological activities of a purified mixture of extracts fromExamples 2.2, 3.6, 3.7, 3.8, 3.9, and 3.10 in a nitric oxide bioassay inmurine macrophages.

FIG. 8: Effects of an extract according to the invention on thesecretion of histamine from compound 48/80-stimulated rat mast cells.

FIG. 9: Mean total colony-forming unit (CFU) values in (a) bladder, and(b) kidney tissues for different experimental groups.

FIGS. 10A-10D: Effect of an embodiment of the invention in anEscherichia coli infection model in an LPS-insensitive strain of mice.The figures show flux cytometry data with (a) markers CD4 vs FoxP3, and(b) TCR vs FoxP3. FIGS. 10A and 10C show untreated tissue while FIGS.10B and 10D show treated tissue.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Extract: An extract, as defined herein, means material obtainedfollowing lysis of one or more bacterial strains. In some cases, theextract is obtained from only one strain while in others the extract isobtained from a mixture of several different strains.

Alkaline lysis: This is a method of lysing bacterial cells under basicconditions.

Lysate: As used herein, this term means an extract of bacteria obtainedfrom a cell lysis procedure.

Filtration: A filtration process, as described herein, means a passageof an extract or a mixture of extracts, through one or more filters suchas microfilters (i.e. microfiltration) or ultrafilters (i.e.ultrafiltration). Such filtration may not necessarily remove 100% of thecomponents it is designed to remove. In some cases, filtration isrepeated in several passes or cycles.

Initial pH: That term means the pH measured at the start of a procedure,such as bacterial lysis or filtration.

Saccharides: A saccharide, as defined herein, includes monosaccharides,disaccharides, as well as larger saccharides such as linear and branchedpolysaccharides. Saccharides also include substituted or chemicallymodified saccharides, such as lipopolysaccharides (LPS) and theirchemically modified variants.

D-amino acids: This term refers to amino acids that exist indextra-rotatory isomeric forms, as opposed to biosynthetically producedL-amino acids, which exist in levo-rotatory isomeric forms.

Racemization: This term indicates at least partial chemical modificationof L-amino acids to D-amino acids.

Medium that avoids the risk of prion-based diseases means a culturemedium used at any stage of the preparation of the extracts that doesnot comprise materials such as serum or meat extracts taken from animalssuch as cows or sheep, or from any other animal that can transmitprion-based diseases. Examples of such media include vegetable-based orsynthetic media and also media using horse serum or media comprisingmaterials taken from animal species that do not transmit prion diseases.Examples of prion-based diseases include, for example, mad cow disease,scrapie, and Creutzfeld-Jacob disease.

A non-animal medium is a medium that does not include components derivedfrom animals. Examples include a vegetable-based (i.e. vegetal) medium,such as a soya medium, and a synthetic medium.

Treatment as used herein means both treatment of current infections, forexample, as well as prevention of or protection from the development ofnew infections, for example.

Subject, as used herein, means any animal subject, including mammaliansubjects, such as humans and domestic mammals.

It is understood that the specific bacterial strains identified hereinand used in the invention may include the strain obtained from theoriginal deposit recited herein or a genetic clone thereof, including astrain that has been re-deposited at a later time with a differentdeposit code name, but which is considered to be genetically the samestrain as the originally deposited version.

All numbers used herein are approximate, taking into account errorsinherent in their measurement, rounding, and significant figures.

Preparation of Extracts

The bacterial extracts of the present invention may be prepared byfermentation followed by heat inactivation and alkaline lysis andfiltration. For each strain, to obtain a sufficient amount of material,the fermentation cultures may be started from a working seed lotfollowed by inoculation into larger fermentation containers.

The media used may be the same for each species. However, supplementarygrowth factors may be introduced to enhance the growth of some species.In some embodiments, a medium that avoids the risk of prion-baseddiseases can be used for growing at least some, or all, strains.Examples include non-animal media such as a vegetable-based medium andsynthetic media. Other examples include a medium that includes horseserum or another animal extract, taken from a species of animal thatdoes not pose a threat of prion diseases, in contrast to strains grownin the presence of bovine serum or meat extracts which can pose suchrisks. In some embodiments, an Ala-Gln dipeptide may be added to themedium. The inventors observed that the Ala-Gln dipeptide, in someembodiments, served as a growth stimulator for the bacterial culture.

After fermentation, the resulting biomass from each strain or from a setof strains may be inactivated by a heat treatment, concentrated, andfrozen. The cellular material may be lysed with hydroxide ions, such asfrom NaOH. In some embodiments, a biomass concentration of 2 to 130 g/Lof bacterial dry weight may be lysed, such as from 20 to 120 g/L, orfrom 5 to 90 g/L, or from 10 to 50 g/L, or from 40 to 90 g/L. (Thebiomass concentration is provided herein as the bacterial dry weight perliter of lysis. The biomass concentration is measured by drying 5 mL ofmaterial in a small porcelain dish at 105° C. until it reaches aconstant mass and then recording the mass in grams per liter.) Forexample, Haemophilus strains may be lysed at a biomass concentration of15-90 g/L, such as from 40 to 90 g/L, such as 40, 50, 60, 70, 80, or 90g/L or smaller ranges bounded by those concentrations (i.e. 40-50,70-90, etc.). Streptococcus strains, for instance, may be lysed at 10 to90 g/L, such as at 10, 20, 30, 40, 50, 60, 70, 80, or 90 g/L or smallerranges bounded by those concentrations. Moraxella strains may be lysedat, for example, 5 to 60 g/L, or at 10-60 g/L, or at 15-40 g/L, such asat 5, 10, 20, 30, 40, 50, or 60 g/L or smaller ranges bounded by thoseconcentrations; Klebsiella strains may be lysed at, for example, 10 to50 g/L, such as 25-50 g/L, or 10, 20, 30, 40, or 50 g/L or smallerranges bounded by those concentrations. Staphylococcus strains may belysed at, for example, 30 to 90 g/L, such as 30, 40, 50, 60, 70, 80, or90 g/L or smaller ranges bounded by those concentrations. Neisseriastrains may be lysed at, for example, 5 to 60 g/L, such as 5, 10, 20,30, 40, 50, or 60 g/L or smaller ranges bounded by those concentrations.

In some embodiments, a hydroxide concentration of 0.01N to 1.2 N may beused for the lysis, such as, from 0.1 to 1N, or from 0.05 N to 0.4 N,such as 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4 N, or smallerranges bounded by those concentrations, or from 0.5 N to 1.0 N, such as0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 N, or smaller ranges bounded by thoseconcentrations. A base concentration may be used so as to achieve ainitial pH of 12 or higher, a pH greater than 12, a pH greater than12.5, or a pH such as from pH 12.0 to pH 13.4 or pH 12.6 to 13.4. Forinstance, for Streptococcus strains, the hydroxide concentration may be0.1-0.7 N, or 0.2-0.5 N, such as 0.2, 0.3, 0.4, or 0.5 N or smallerranges bounded by those concentrations. For Moraxella or Haemophilusstrains, it may be 0.05-0.7 N, or 0.15-0.5 N, such as 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, or 0.5 N or smaller ranges bounded by thoseconcentrations. For Klebsiella or Staphylococcus strains, it may be0.1-0.7 N, or 0.15-0.4 N such as 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4 N orsmaller ranges bounded by those concentrations.

The lysis temperature may be from 30 to 60° C., such as from 30-40° C.,or from 35-40° C., such as 37° C. The time of lysis may vary from 20hours or from 40 hours to several days, such as 2, 3, 4, 5, 6, 7, 8, 9,or 10, or even 15 days. For instance, for Haemophilus, Streptococcus,Moraxella, and Staphylococcus strains, a time of 5-9 days may beemployed, and a time of 7-10 days for Klebsiella and Neisseria strains.In some embodiments, lysis temperatures of 30-40° C., or 35-40° C., suchas 37° C., may be employed for each of the strains and the lysis mayoccur over a period of 72 to 210 hours (3-9 days), such as 3 days, 4, 5,6, 7, 8, and 9 days or ranges of hours or days bounded by those times(e.g., 3-4 days, 8-9 days, etc.). It is understood that these ranges oftime include any fractional number of days, hours, or minutes, therein.

In some embodiments, when using more than one strain of the samebacterial genus, the strains may be lysed together or separately. Thestrains may be mixed before or after lysis.

The extracts may be purified by centrifugation and/or filtration. Forexample, lysates may be centrifuged at 9000× gravity, followed by one ormore rounds of filtration on a 0.2 micron filter. In some cases,successive rounds of filtration on larger pore filters followed byfiltration on a 0.2 micron filter may be used. Ultrafiltration methodsmay also be employed in order to help extract soluble materials from theextract, for example, recirculating the ultrafiltration permeate forfurther microfiltration.

In some embodiments, a tangential flow filtration (TFF) method may beused to filter the extracts and to extract solubilized molecules fromlarger cellular debris. (See FIG. 1.) (See, e.g., SeparationsTechnology, Pharmaceutical and Biotechnology Applications, Wayne P.Olson, Editor. Interpharm Press, Inc., Buffalo Grove, Ill., U.S.A., p.126 to 135-ISBN:0-935184-72-4.) At the beginning of such a process, adiluted bacterial lysate may be stored in a first tank. Amicrofiltration (MF) loop is started, and the product is pumped. Theresulting MF retentate is recycled, while the MF permeate is transferredto a second tank.

After reaching a suitable degree of concentration, an ultrafiltration(UF) loop may be started. The UF permeate may be recirculated back tothe first tank for continuous extraction of solubilized compounds fromthe lysate while the UF retentate is stored in the second tank. Duringthe continuous extraction, the volumes in tanks 1 and 2 may be adjustedby regulation of flow rates of the microfiltration and ultrafiltrationpermeates.

Several such extraction cycles may be performed, either with TFF oranother filtration method. In embodiments that use TFF, at the end ofthe last cycle, the ultrafiltration loop may be shut down and themicrofiltration loop may be run alone and the MF permeate transferred totank 2.

The microfiltration loop may be fitted with filters of 1.2 microns to0.1 microns, such as filters of 0.65 to 0.2 microns, or 0.45 microns.The cross-flow may be between 1000 Liters/hours m² (LHM) and 3000 LHM,such as between 1500 and 2500 LHM, or 2000 LHM with a trans-membranepressure (TMP) of 0.6 to 2 bars, such as between 0.8 and 1.5 bars, or1.0 bar. The ultrafiltration loop may be fitted with filters of from 10KDa to 1000 KDa, such as from 10 KDa to 100 KDa, or from 10 KDa to 30KDa, or from 30 KDa to 100 KDa. The cross-flow may be between 30 LHM and1000 LHM, such as between 20 and 500 LHM with a TMP of 0.2 to 1.5 bars,such as between 0.4 and 1.2 bars, or 0.5 bar.

Between 5 and 20 diafiltration volumes may be used to extractsolubilized compounds from bacterial cell walls. In some embodiments,between 8 and 15 volumes are used. Hence, for example, in someembodiments, between 5 and 15 cycles of filtration may be used, in somecases between 8 and 15 cycles, such as 8, 9, 10, 11, 12, 13, 14, or 15cycles.

Following filtration, the extracts may be further diluted, concentratedor centrifuged, if desired. Purification steps may also be included toremove particulate matter from the extracts. For instance, a furthermicrofiltration using a smaller pore filter may be performed, such as a0.2 micron filter. After filtration, the yield of solubilized proteinsmeasured by Lowry may be more than 50%, or may be more than 60%, or maybe 50 to 90%, or may be 60-90%, for example. Following filtration, theextract may be lyophilized prior to formulating it for use.

In some embodiments of the invention, a group of lysis conditions may bechosen an applied to one or more bacterial strains. From 40 to 90 g/L,or 40, 50, 60, 70, 80, or 90 g/L of Haemophilus influenzae NCTC 8467 maybe lysed for 72-200 hours, such as 72, 96, 120, 150, or 200 hours. From10 to 95 g/L of one or more Streptococcus strains, such as 10, 20, 40,60, 80, 90, or 95 g/L may be lysed for 72-210 hours, such as 72, 96,120, 150, or 200 hours. From 15 to 80 g/L of one or more Diplococcusstrains, such as 15, 20, 30, 40, 50, 60, 70, or 80 g/L may be lysed for72-210 hours, such as 72, 96, 120, 150, or 200 hours. From 10 to 50 g/Lof one or more Klebsiella strains, such as 10, 15, 20, 25, 30, 35, 40,45, or 50 g/L may be lysed for 72-210 hours, such as 72, 96, 120, 150,or 200 hours. From 5 to 60 g/L of one or more Neisseria strains, such as5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 g/L may be lysed for72-210 hours, such as 72, 96, 120, 150, or 200 hours. From 30 to 90 g/Lof one or more Staphilococcus strains, such as 30, 40, 50, 60, 70, 80,or 90 g/L may be lysed for 72-210 hours, such as 72, 96, 120, 150, 200hours. In each of those embodiments, the lysis may be conducted at35-40° C., such as at 37° C. Further, either “moderate” or “strong”lysis conditions may be used for each strain forming the extract or agroup of similar strains. As used herein, “moderate” lysis conditionsrefer to a hydroxide ion concentration of 0.05 to 0.4 N, such as 0.1,0.2, 0.3, or 0.4 N together with the biomass, time, and temperatureparameters given just above for each type of strain (e.g. 35-40° C.,40-90 g/L of biomass and 72-200 hours for Haemophilus influenzae NCTC8467). As used herein, “strong” lysis conditions refer to a hydroxideion concentration of 0.5 to 1N, such as 0.5, 0.6, 0.7, 0.8, 0.9 and 1N,together with the time, temperature, and biomass parameters for eachstrain given just above. In some embodiments, both strong and moderatelyses may be conducted, with the resulting products mixed together.

In some embodiments of the invention, the extract may be obtained frommore than one bacterial strain, such as from at least one gram negativeand at least one gram positive strain. Bacterial strains from the sameor different species may be mixed before or after lysis. In someembodiments, strains may be mixed, for example, to obtain 1-40% volumeof each in the mixture or 15-30% by volume of each genus of bacteria. Insome embodiments, the extract may comprise lysis products from, forexample, 2, 3, 4, 5 or 10 different genera of bacteria. For instance amixture of 5 genera may comprise Haemophilus, Moraxella, Klebsiella,Staphylococcus, Streptococcus strains, including Streptococcussanguinis, pyogenes, and pneumoniae. Some Streptococcus pneumoniaestrains are also known as, for example, Diplococcus strains. In somesuch 5 genus embodiments, the mixture may contain from 5 to 15% ofHaemophilus by volume, such as 7-10%, or 7, 8, or 9%; from 5 to 15% ofDiplococcus by volume, such as 7-10% or 7, 8, or 9%; from 5-20% ofStreptococcus by volume, such as 7-15%, or 8, 9, 10, 11, or 12%; from 10to 30% of Klebsiella by volume, such as 15-25%, or 16, 17, 18, 19, 20,21, 22, 23, or 24%; from 10 to 30% of Staphylococcus by volume, such as15-25%, or 16, 17, 18, 19, 20, 21, 22, 23, or 24%; and from 20 to 40% ofNeisseria by volume, such as 25-35%, or 26, 27, 28, 29, 30, 31, 32, 33,or 34%.

Chemical Properties of Bacterial Extracts

Some embodiments according to the present invention may contain, forexample, 5-75 mg/mL of proteins, or 10-65 mg/mL, or 20-45 mg/mL, or 5-40mg/mL, or 5-20 mg/mL, or 5-10 mg/mL, or 6-8 mg/mL of proteins or a rangestarting or ending from 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70 or 75 mg/mL; 1.5 to 2.5 mg/mL of free amino acids (A.A.), or1.5 to 2 mg/mL, or 2 to 2.5 mg/mL of free A.A., or a range starting orending from 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5mg/mL of free A.A., calculated from glutamic acid (147.1 g/mol); and 0.3to 4.5 mg/mL of polysaccharides and monosaccharides, or 0.3 to 4 mg/mL,or 0.4 to 4 mg/mL, or 0.5 to 3.5 mg/mL, or 0.6 to 3 mg/mL or 0.3 to 1mg/mL or a range starting or ending from 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mg/mL of polysaccharidesand monosaccharides, such as, e.g. 0.4 to 0.5 mg/mL. For example, someembodiments contain about 6 to 8 mg/mL of proteins, 1.5 to 2.5 mg/mL offree amino acids (A.A.), calculated from glutamic acid (147.1 g/mol)and/or about 0.4 to 0.5 mg/mL of polysaccharides and monosaccharides.Protein concentration is measured by the Lowry assay in accordance withmethod 2 of European Pharmacopoeia 2.5.33. The sugar concentration isassayed after acid hydrolysis and derivatization according to D. Herbertet al., Meth. Microbiol. 5B: 266 et seq. (1971). The glutamate (glutamicacid) concentration is measured by converting amino acids to isoindolederivatives and measuring absorbance at 340 nm, according to Roth M.,Fluorescence reaction for amino acids, Anal. Chem., 43, 880-882, (1971).

In some embodiments, the concentration of LPS equivalents based on alimulus amoebocyte lysate (LAL) chromogenic test is less than 1000ng/ml, or less than 500 ng/ml, less than 200 ng/ml, or less than 100ng/ml.

Lysis of bacteria according to the present invention may result inpartial hydrolysis of proteins as well as deamination, deamidation, andpartial racemization of amino acids from L to D. In one analytical studyof an extract according to the invention, peaks representing D-asparticacid, D-glutamic acid, D-serine, D-methionine, D-histidine, D-alanine,D-arginine, D-phenylalanine, D-tyrosine, D-leucine, and D-lysine wereeach observed. The percentage of D-amino acids of those species in thatstudy ranged from 3% to 40%. Hence, some embodiments of the inventionallow for racemization of one or more of serine, threonine, histidine,alanine, arginine, tyrosine, phenylalanine, leucine, and lysine, such asall of the above amino acids, or any selection of more than one but lessthan all of the above amino acids, such as, for example, alanine,phenylalanine and lysine. In some embodiments, at least 10% of one ormore of the above amino acids may become racemized from D to L. In otherembodiments, at least 40% of one or more of the above amino acids maybecome racemized.

Lysis of bacteria according to the present invention may result in adiminution of the molecular weight of component molecules from 0 to 300kDa to 0 to 100 kDa, or 0 to 60 kDa due to hydrolysis.

Biological Activities of Bacterial Extracts

Extracts according to the invention may be effective to treat patientssuffering from or at risk of developing medical conditions such asrespiratory disorders and allergic reactions or conditions. Extractsaccording to the invention may be effective to treat, for example, upperand lower respiratory infections, atopic dermatitis, nasopharyngitis,sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis,laryngopharyngitis, influenza, pneumonia, bronchopneumonia, bronchitis,lower respiratory infections, allergic rhinitis, allergic asthma,rhinitis, nasopharyngitis, pharyngitis, sinusitis, tonsillitis,laryngitis, laryngotracheitis, bronchitis, obstructive pulmonary diseasewith acute lower respiratory infection, and obstructive pulmonarydisease with acute exacerbation.

Biological activity of extracts may be determined by several assays. Forexample, a peripheral blood mononuclear cell (PBMC) assay tests theproduction of the cytokine IL-6 from PBMC's and can screen for theability of an extract to stimulate the immune system. For example, insome embodiments, the in vitro IL-6 concentration measured insupernatants of PBMCs stimulated with the extracts of the inventionranged from 2000 pg/ml to 70,000 pg/ml, 2000 pg/ml to 50,000 pg/ml, 2000pg/ml to 30,000 pg/ml, 2000 pg/ml to 20,000 pg/ml, 2000 pg/ml to 10,000pg/ml, or 5000 pg/ml to 70,000 pg/ml, 5000 pg/ml to 50,000 pg/ml, 5000pg/ml to 30,000 pg/ml, 5000 pg/ml to 25,000 pg/ml, or 5000 pg/ml to10,000 pg/ml, or 15,000 pg/ml to 25,000 pg/ml. When LPS was used as anagonist control (at 0.01 μg/ml), the values obtained ranged, dependingfrom the donors, from 5,000 pg/ml to 70,000 pg/ml.

A murine nitric oxide (NO) test measures production of NO by murinemacrophages, which also indicates immune stimulation. For example,macrophages produce NO in order to kill invading bacteria. In someembodiments, in vitro nitrous oxide (NO) activity for embodiments of thepresent invention tested at concentrations ranging from 0.001 mg/ml to10 mg/ml of soluble dry weight provided maximal responses ranging from 3μM to 100 μM nitric oxide, or 3 μM to 90 μM, 3 μM to 80 μM, 3 μM to 70μM, 3 μM to 60 μM, 3 μM to 50 μM, 3 μM to 40 μM, 3 μM to 30 μM, 3 μM to20 μM, 3 μM to 10 μM, or 5 μM to 80 μM, 5 μM to 60 μM, 5 μM to 40 μM, 5μM to 20 μM, or 10 μM to 80 μM, 10 μM to 70 μM, 10 μM to 50 μM, 10 to 30μM, or 10 μM to 15 μM, or ranges beginning or ending from 3, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100μM.

Activities observed on human peripheral blood mononuclear cells andmurine macrophages in vitro may depend on variables such as the amountof bacterial dry weight biomass to be lysed, i.e. the “startingmaterial” for lysis, the duration of the alkaline lysis, and the initialpercentage of NaOH or initial pH used in the lysis.

Combination of in vitro activity tests such as PBMC and NO withdetermination of LPS concentration such as by LAL also may provideinformation concerning the balance of activity vs. toxicity risk for agiven bacterial extract.

The extracts of this invention may also be active against aerosolinfluenza virus infection, such as against A/PR/8/34 (H1N1) infection.In one study, for example, an extract according to the present inventionwas able to confer complete immunoprotection in mice at a dose of 10mg/mouse, as judged by mortality, lung virus titration, clinicalsymptoms, and antibody titers. In contrast, only 70% of control animalssurvived infection.

As another example, the survival rate 13 days after challenge of atleast 8 mice having wild-type LPS sensitivity with Salmonellathyphimurium, is at least 60% when those mice are first treated for 10days with effective amounts of some embodiments of the presentinvention. The dose of Salmonella thyphimurium for the challenge may bechosen such that untreated mice or mice treated with a water or blankformulation control containing excipients but no extract have a survivalrate of 60% or less, such as 50% or less. In some cases, the survivalrate for the extract-treated mice is at least 70%, at least 80%, atleast 80%, at least 90%, or at least 95%.

Furthermore, embodiments of this invention may also inhibit thesecretion of histamine by compound 48/80-stimulated mast cells to astatistically significant degree, as shown in detail further below. Forexample, embodiments of the invention may exhibit an IC-50 value in acompound 48/80-stimulated mast cell assay of, for example, between0.0005 and 0.01 mg/mL, such as between 0.0005 and 0.005 mg/mL, orbetween 0.001 and 0.01 mg/mL, or between 0.002 and 0.008 mg/mL, orbetween 0.004 and 0.006 mg/mL, for example.

Compositions Comprising the Bacterial Extracts

The lyophilized extract mixture may be formulated in a number ofdifferent ways for eventual administration. For example, oral tablets,capsules, pills, may be prepared, as well as liquid formulations oraerosols. Formulations for infusion or injection may also be prepared.Embodiments of this invention can be formulated, for example, as soliddosage forms or liquid dosage forms. Exemplary solid dosage forms mayinclude, for example, a tablet, e.g. coated tablet, chewable tablet,effervescent tablet, sublingual tablet, granulates, powder, or acapsule) containing the extract, and optionally, one or more nutritionaland/or dietary supplements. Solid dosage forms may also containdiluents, fillers, and/or other excipients. Other excipient componentsmay be added such as preservatives, colorants, flavourings, andsweeteners. It is also possible to prepare powder or granulateformulations. Liquid dosage forms as solutions, syrups, suspensions, ordrops can also be utilized for the oral route.

WORKING EXAMPLES Example 1 Bacterial Cultures Example 1.1 Culture ofHaemophilus Influenzae NCTC 8467 Initial Culture Conditions

Culture media was prepared by dissolving in purified water the followingcomponents: Sodium chloride: 3 g/L; Sodium monohydrogen phosphate: 2g/L; Sodium acetate: 0.5 g/L; Soya peptone 40 g/L; Glucose: 6 g/L;Inosine: 0.1 g/L; Calcium chloride: 0.02 g/L; Potassium chloride: 0.1g/L; Sodium bicarbonate: 0.6 g/L; Sodium pyruvate: 0.06 g/L; Metalsolution (copper sulfate: 3 mg/l; iron chloride: 830 mg/l; zinc sulfate:860 mg/l; sulfuric acid: 1.1 mg/L): 0.5 mL/L; Hemin: 25 mg/l; NADH(β-nicotinamide adenine dinucleotide disodium salt reduced trihydrate)25 mg/l. After dissolution, the pH was adjusted to 7.2. Aftersterilizing the media, small Erlenmeyer flasks were individuallyinoculated with the content of frozen vials (containing 1.5 mL of frozenbacteria) and incubated at 37° C. for 8 hours. Then aliquots of thisculture were transferred to larger Erlenmeyer flasks containing 150 mLof culture media, and incubated again in the same conditions. Anotherfermentation step with 1000 mL of culture media was performed in thesame conditions but with 50 mg/L of hemin and 50 mg/L of NADH addedbefore inoculation (OD at 700 nm for the 10 ml culture 1 after 10 hours:3.7, for the 1000 ml culture after 11 hours: 13.5.). Then, the entire1000 ml culture content was transferred to prefermenters.

Culture Conditions in Prefermenters

Culture media was prepared by dissolving in purified water the followingcomponents: Sodium chloride: 3 g/L; Sodium monohydrogen phosphate 2 g/L;Sodium acetate: 0.5 g/L; Soya peptone 40 g/L; Glucose 6 g/L; Inosine 0.1g/L; Calcium chloride 0.02 g/L; Potassium chloride 0.1 g/L; Sodiumbicarbonate 0.6 g/l; Sodium pyruvate 0.06 g/l; Metal solution: 0.5 mL/L;Hemin: 1 mg/L; NADH 10 mg/L, Polypropylene glycol: 0.06-0.10, mL/L. Theincubation temperature was regulated at 30° C., with stirring andaeration. The pH was not regulated during the culture. After 13 hours, 2prefermenters were transferred to a fermenter (OD at 700 nm culture 1after 6 hours: 1.53; culture 2 after 8.5 hours: 1.90). The cultures ofthe prefermenters were transferred under sterile conditions intofermenters.

Culture Conditions in Fermenters

Culture media was prepared by dissolving in purified water the followingcomponents: Sodium chloride: 3 g/l, Sodium monohydrogen phosphate: 2g/L; Sodium acetate 0.5 g/L, Soya peptone 40 g/L; Inosine 0.1 g/L;Calcium chloride 0.02 g/L; Potassium chloride 0.1 g/L, Sodiumbicarbonate 0.6 g/L; Sodium pyruvate:0.06 g/L; Metal solution:0.5 mL/L;Hemin 1.5 mg/l; NADH: 15 mg/l; Polypropylene glycol: 0.02-0.04 mL/L.

After sterilization, 15 g/L glucose was added to the culture. Theincubation temperature was regulated at 35° C., with stirring andaeration. The pH was regulated at 6.8 during the culture. After 8 hours,the cultures (OD at 700 nm, culture 1 after 7.25 hours: 3.69; culture 2after 8.75 hours: 3.55) were inactivated by heat treatment at 90 to 100°C. and transferred to a harvest Tank. Once inactivated, the cultureswere transferred to a centrifuge in order to separate the biomass fromthe culture medium and concentrate. Harvested biomass was stored in atank connected to a centrifuge. The retentate of centrifuge (10001/h)was recycled to a storage tank whereas the permeate was evacuated. Thebiomass was concentrated and then harvested in a sterile tank. After3.25 hours, 31,768 g of biomass were harvested. OD of the concentratedbiomass was 237.4 at 700 nm. The biomass was divided into a series ofaliquots containing 425 g of dry weight biomass. The aliquots were thenfrozen at −15° C.

Example 1.2 Staphylococcus Cultures

Culture conditions in Erlenmeyer Flasks

Culture media for Staphylococcus aureus 049 (StAu 049), Staphylococcusaureus I-050 (StAu 050), Staphylococcus aureus I-051 (StAu 051),Staphylococcus aureus I-052 (StAu 052), Staphylococcus aureus I-053(StAu 053) and Staphylococcus aureus I-054 (StAu 054) was prepared bydissolving in purified water the following components: Sodium chloride 2g/L; Sodium monohydrogen phosphate 2 g/L; Sodium acetate 0.5 g/L; Soyapeptone 40 g/L; Glucose 6 g/L. Then 0.012 L of media was inoculated with1.5 mL of frozen bacteria. The culture was incubated at 37° C. for 7hours under stirring at 180 rpm and pH 6.9. Successive culture stepsfrom 12 to 1000 mL were performed.

Culture Conditions in Prefermenters

The same media as the preceding step was prepared for prefermenters, butwith addition of polypropylene glycol 0.06-0.10 mL/L. Media wassterilized in situ at 123° C. for 30 min. 1000 ml of culture from theprevious step was transferred into prefermenters with stirring andaeration. The incubation temperature was regulated at 37±2° C. The pHwas not regulated during the culture. After 6 hours, the 2 prefermenterswere transferred to fermenters.

Culture in Fermenters

Culture media for StAu 049 was prepared by dissolving in purified waterthe following components: Sodium chloride 2 g/L; Sodium monohydrogenphosphate 2 g/L; Sodium acetate 0.5 g/L; Soya peptone 40 g/L;Polypropylene glycol 0.04 mL/L. The media was sterilized in situ.Glucose (14 g/L) was added to the culture. The incubation temperaturewas regulated at 37° C. with stirring and aeration. The pH was regulatedat 6.4±0.5. After 7 hours, the cultures were inactivated by heattreatment at 90 to 100° C. and transferred to a harvest tank. Thebiomass was then separated from the culture media by centrifugation. Thebiomass was divided into a series of aliquots containing a certainamount of dry weight biomass.

The biomass dry weight in each aliquot was: StAu 049: 327 g, StAu 050:297 g, StAu 051: 375 g, StAu 052: 363 g, StAu 053: 446 g and StAu 054:365 g.

Example 1.3 Klebsiella Cultures Initial Culture Conditions

Culture media for Klebsiella pneumoniae NCTC 5050 (Klba 5050),Klebsiella pneumoniae NCTC 5056 (Klba 5056) and Klebsiella pneumoniaeNCTC 204 (Klba 204) was prepared by dissolving in purified water thefollowing components: Sodium chloride 2 g/L; Sodium monohydrogenphosphate 2 g/L; Sodium acetate 0.5 g/L; Soya peptone 40 g/L; Glucose 6g/L. Then 0.012 L of media was inoculated with 1.5 mL of frozenbacteria. The culture was incubated at 37° C. for 10 hours with stirringand an initial pH set at 6.9. Successive culture steps from 0.012 to 1.0Liters were performed.

Culture Conditions for Klba 5050 in Prefermenters

The same media as the previous step was prepared for prefermenters, butwith addition of polypropylene glycol 0.06 mL/L. One liter of culturefrom the previous step was transferred to prefermenters. The incubationtemperature was regulated at 37° C. with stirring and aeration. The pHwas not regulated during the culture. After 6 hours, the twoprefermenters were transferred to a fermenter.

Culture Conditions in Fermenters

Culture media for Klba 5050 was prepared by dissolving in purified waterthe following components: Sodium chloride 2 g/L; Sodium monohydrogenphosphate 2 g/L; Sodium acetate 0.5 g/L; Soya peptone 40 g/L andpolypropylene glycol 0.02 mL/L. The media was sterilized in situ and 21g/L glucose was added. The incubation temperature was regulated at 37°C. with stirring and aeration. The pH was regulated at 6.6 during theculture. After 8 hours, the cultures were inactivated by heat treatmentat 90 to 100° C. and transferred to a harvest tank. The biomass was thenseparated from the culture media by centrifugation. Biomass dry weightin each aliquot was: Klba 5050: 393 g, Klba 5056: 455 g and Klba 204:440 g.

Example 1.4 Moraxella Catarrhalis Cultures Initial Culture Conditions

Culture media for Moraxella catarrhalis NCTC 3622 (NeCa 3622); Moraxellacatarrhalis NCTC 3625 (NeCa 3625) and Moraxella catarrhalis I-045 (NeCa045) was prepared by dissolving in purified water the followingcomponents: Sodium chloride: 3 g/L; Sodium monohydrogen phosphate: 2g/L; Sodium acetate: 0.5 g/L; Soya peptone: 40 g/L; Starch: 0.1 g/L;Inosine: 0.1 g/L; Calcium Chloride: 0.02 g/L; Potassium Chloride: 0.1g/L; Sodium bicarbonate: 0.6 g/L; Sodium pyruvate: 0.06 g/L; Metalsolution: 0.5 mL/L (Metal Solution composition: Copper Sulfate 3 mg/L;iron Chloride: 830 mg/L; zinc sulfate: 860 mg/L; sulfuic acid: 1.1mg/L); Dipeptide ALA-GLN (200 mg/mL in 0.9% NaCl Solution): 10 mg/L (forfirst culture step 50 mg/L; for 1 Liter culture step 10 mg/L). Then0.012 L of media was inoculated with 1.5 mL of frozen bacteria. Theculture was incubated at 37° C. for 10 hours with stirring. The initialpH was set at 7.2. Successive culture steps from 0.012 to 1.0 L wereperformed in the same conditions.

Culture in Prefermenters

The same media as the previous step was prepared for prefermenters, butwith addition of polypropylene glycol 0.06-0.10 mL/L and concentrationof ALA-GLN adjusted to 4 mg/L. Media was sterilized in situ. One literof culture from the previous step was transferred to prefermenters. Theincubation temperature was regulated at 33° C. with stirring andaeration. The pH was not regulated during the culture. After 10 hours,the 2 prefermenters were transferred to fermenters.

Culture Conditions in Fermenters

The same media as the Erlernmeyer step was prepared for prefermenters,but with addition of polypropylene glycol 0.06 mL/L and without ALA-GLNor glucose. The incubation temperature was regulated at 33° C. withstirring and aeration. The pH was not regulated during the culture.After 10.5 hours, the cultures were inactivated by heat treatment at 90to 100° C. and transferred to a harvest tank. The biomass was thenseparated from the culture media by centrifugation. The biomass dryweight in each aliquot was: NeCa 3622: 361 g, NeCa 3625: 351 g and NeCa045: 223 g.

Example 1.5 Streptococcus Cultures Initial Culture Conditions

Culture media for Streptococcus pneumoniae NCTC 7465 (StPn 7465),Streptococcus pneumoniae NCTC 7466 (StPn 7466), Streptococcus pneumoniaeNCTC 7978 (StPn 7978), Streptococcus pneumoniae NCTC 10319 (StPn710319), Streptococcus sanguinis I-046 (StSa 046), Streptococcussanguinis I-047 (StSa 047), Streptococcus sanguinis I-048 (StSa 048) andStreptococcus Pyogenes NCTC 8191 (StPy 8191) was prepared by dissolvingin purified water the following components: Sodium chloride: 2 g/L;Sodium monohydrogen phosphate: 2 g/L; Sodium acetate: 0.5 g/L; Soyapeptone: 40 g/L; Glucose: 6 g/L; Horse Serum: 50 mL/L. Aftersterilisation, 0.012 L of media was inoculated with 1.5 mL of frozenbacteria. The culture was incubated at 37° C. for 14 hours withstirring. Then, 10 mL of this culture was transferred to flaskscontaining 150 mL of culture media and incubated again in the sameconditions for 10 hours. A final step was performed under the sameconditions in a larger flask containing 1000 mL of culture media with 20mL/L Horse Serum before being added to the prefermenter.

Culture in Prefermenters

The same media as the previous step was prepared for prefermenters, withaddition of polypropylene glycol 0.06 mL/L and a concentration of HorseSerum of 8 mL/L. One liter of the previous culture was transferred totwo prefermenters. The incubation temperature was regulated at 30° C.with stirring. The pH was not regulated during the culture. After 14hours, the 2 prefermenters were transferred to a fermenter.

Culture in Fermenters

The same media as the Erlenmeyer step was prepared for prefermenters,but with addition of polypropylene glycol 0.06 mL/L and a Horse Serumconcentration of 1.2 mL/L. 15.75 kg of glucose was added during theculture. The incubation temperature was regulated at 37° C. withstirring. The pH was regulated at 6.4 with concentrated KOH. After 9.25hours, the cultures were inactivated by heat treatment at 90 to 100° C.and transferred to a harvest tank. The biomass was then separated fromthe culture media by centrifugation. StSa 046, StSa 047, StSa 048 andStPy 8191 were aerated with sterile air and did not need addition ofHorse serum for the culture. The biomass dry weight in each aliquot was:StPn 7465: 134 g, StPn 7466: 142 g StPn 7978: 134 g StPn 10319: 153 gStSa 046: 246 g, StSa 047: 232 g, StSa 048: 353 g and StPy 8191: 269 g.

Example 2 Bacterial Lysis HaIn 8467 Example 2.1

Aliquots of HaIn 8467 from Fermentation Example 1.1 of Bacterial biomasswere thawed at room temperature and diluted with a saline solution (8g/L NaCl) to reach 79 g/L dry weight. Alkalinization at 0.2 M NaOH wasperformed. The lysis was incubated for 120 hours at 35-40° C. undercontinuous stirring. During the lysis, the pH was monitored so as not todecrease by more than 0.5 pH units. Results. 82.2 mg/mL of solubilizeddry weight (SDW) and 32.4 mg/mL of proteins (Prot) and 6.2 mg/mL oftotal amino acids (A.A) calculate in glutamic acid (147.1 g/mol),measured by OPA, 2.40 mg/mL of reducing sugars measured by OPA(Carbohydrates).

Protein concentration (Prot) was measured by a Lowry assay (see EuropeanPharmacopoeia 2.5.33, under “total protein—method 2”). The total freeamino acid concentration (A.A) was measured by converting amino acids toisoindole derivatives and measuring absorbance at 340 nm, according toRoth M., Fluorescence reaction for amino acids, Anal. Chem., 43,880-882, (1971). Results are expressed in equivalents of glutamic acid.The sugar (Carbohydrates) concentration was assayed after acidhydrolysis and derivatization according to D. Herbert et al., Meth.Microbiol. 5B: 266 et seq. (1971).

HaIn 8467 Example 2.2

Biomass according to Example 1.1 was diluted to 58.2 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 5 days at35-40° C. under continuous stirring. (Soluble Dry Weight (SDW): 70.0mg/ml; Lowry Protein (Prot): 30.0 mg/ml; Amino Acids (A.A): 6.0 mg/ml;Carbohydrates: 2.60 mg/ml.) NOx production in mg of active dryweight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 18.7μM, C2: 20.8 μM, C3: 12.1 μM.

HaIn 8467 Example 2.3

Biomass according to Example 1.1 was diluted to 20 g/L. Alkalinizationat 0.045 M NaOH was performed. The lysis was incubated for 5 days at35-40° C. under continuous stirring. (SDW: 29.99 mg/ml. Prot: 4.8 mg/ml;Carbohydrates: 0.2 mg/ml.)

HaIn 8467 Example 2.4 (OP0662L)

Biomass according to Example 1.1 was diluted to 12.5 g/L. Alkalinizationat 0.05 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 11.2 mg/ml; Prot: <0.2 mg/ml;A.A: 2.0 mg/ml; Carbohydrates: 0.4 mg/ml.). D-amino acid percentage: 15%D-Ala, 9% D-Leu, 45% D-Ser, 21% D-Asx, 15% D-Met, 11% D-Phe, 9% D-Glx.

HaIn 8467 Example 2.5

Biomass according to Example 1.1 was diluted to 127 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 157.2 mg/ml; Prot: 86 mg/ml;A.A: 20.0 mg/ml; Carbohydrates: 4.0 mg/ml.) D-amino acid percentage: 22%D-Ala, 11% D-Leu, 54% D-Ser, 41% D-Asx, 35% D-Met, 32% D-Phe, 29% D-Glx,6% D-Tyr. NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1),0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 9.1 μM, C2: 18.5 μM, C3: 3.1 μM.

HaIn 8467 Example 2.6

Biomass according to Example 1.1 was diluted to 12.5 g/L. Alkalinizationat 0.05 M NaOH was performed. The lysis was incubated for 3 days at35-40° C. under continuous stirring. (SDW: 10.8 mg/ml; Prot: less than0.5 mg/ml; A.A: 2.0 mg/ml; Carbohydrates: 0.4 mg/ml) D-amino acidpercentage: 10% D-Ala, 9% D-Leu, 56% D-Ser, 22% D-Asx, 16% D-Met, 13%D-Phe, 11% D-Glx. NOx production in mg of active dry weight/mL, 0.02mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 20.2 μM, C2: 26.7μM, C3: 4.3 μM.

HaIn 8467 Example 2.7

Biomass according to Example 1.1 was diluted to 127 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 3 days at35-40° C. under continuous stirring. (SDW: 168.8 mg/ml; Prot: 90 mg/ml;A.A: 22 mg/ml; Carbohydrates: 4.2 mg/ml) D-amino acid percentage: 36%D-Ala, 8% D-Leu, 9% D-Ser, 44% D-Asx, 42% D-Met, 37% D-Phe, 37% D-Glx,37% D-Tyr.

HaIn 8467 Example 2.8

Biomass according to Example 1.1 was diluted to 12.5 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 47.2 mg/ml; Prot: less than0.2 mg/ml; A.A: 4.0 mg/ml; Carbohydrates: 0.2 mg/ml) D-amino acidpercentage: 3% D-Ala, 13% D-Leu, 54% D-Ser, 45% D-Asx, 43% D-Met, 42%D-Phe, 41% D-Glx. NOx production in mg of active dry weight/mL, 0.02mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 7.5 μM, C2: 16.0 μM,C3: 8.6 μM.

HaIn 8467 Example 2.9

Biomass according to Example 1.1 was diluted to 127 g/L. Alkalinizationat 0.05 M NaOH was performed. The lysis was incubated for 196 hours at35-40° C. under continuous stirring. D-amino acid percentage: 14% D-Ala,5% D-Asx, 11% D-Met, 5% D-Glx.

StPy 8191 Example 2.10

One aliquot of StPy 8191 from Example 1.5, containing 269 g of bacterialmaterial was thawed at room temperature and was diluted with a salinesolution (8 g/L NaCl) to reach 59.8 g/L dry weight. Alkalinization at0.2 M NaOH was performed. Then, the lysis was incubated for 192 hours at35-40° C. under continuous stirring. During the lysis, the pH wasmonitored so as not to decrease by more than 0.5 pH units. (SDW: 61.92mg/mL; Prot: 31.68 mg/mL; A.A.: 7.2 mg/mL; Carbohydrates: 7.2 mg/mL).D-amino acid percentage: 22% D-Ala, 11% D-Leu, 54% D-Ser, 41% D-Asx, 35%D-Met, 32% D-Phe, 29% D-Glx, 6% D-Tyr. NOx production in mg of activedry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1:4.9 μM, C2: 11.8 μM, C3: 6.7 μM.

StSa 046 Example 2.11

Biomass according to Example 1.5 was diluted to 30 g/L. Alkalinizationat 0.022 N NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring.

StPy 8191 Example 2.12

Biomass according to Example 1.5 was diluted to 100 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 120.2 mg/ml; Prot: 45.6mg/ml; A.A: 15.2 mg/ml; Carbohydrates: 2.8 mg/ml) D-amino acidpercentage: 35% D-Ala, 13% D-Leu, 57% D-Ser, 44% D-Asx, 40% D-Met, 39%D-Phe, 43% D-Glx.

StPy 8191 Example 2.13

Biomass according to Example 1.5 was diluted to 12.7 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 2 days at 35-40°C. under continuous stirring. (SDW: 12.5 mg/ml; Prot: <0.2 mg/ml; A.A:0.8 mg/ml; Carbohydrates: 0.1 mg/ml) D-amino acid percentage: 24% D-Ala,13% D-Leu, 52% D-Ser, 28% D-Asx, 8% D-Met, 8% D-Phe, 23% D-Glx, 6%D-Tyr.

StPy 8191 Example 2.14

Biomass according to Example 1.5 was diluted to 100 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 41.4 mg/ml; Prot: 3.2 mg/ml; A.A: 4mg/ml; Carbohydrates: 1.5 mg/ml.)

StPy 8191 Example 2.15

Biomass according to Example 1.5 was diluted to 12.7 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 52.5 mg/ml; Prot: 0.8 mg/ml;A.A: 3.2 mg/ml; Carbohydrates: 0.6 mg/ml.)

StPy 8191 Example 2.16

Biomass according to Example 1.5 was diluted to 100 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 147.2 mg/ml; Prot: 53.6mg/ml; A.A: 24.8 mg/ml; Carbohydrates: 5.4 mg/ml) D-amino acidpercentage: 44% D-Ala, 26% D-Leu, 11% D-Ser, 45% D-Asx, 43% D-Met, 42%D-Phe, 46% D-Glx. NOx production in mg of active dry weight/mL: 0.02mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 9.7 μM, C2: 17.4 μM,C3: 2.5 μM.

StPy 8191 Example 2.17

Biomass according to Example 1.5 was diluted to 12.7 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 14.7 mg/ml; Prot: 0 mg/ml;A.A: 0.8 mg/ml; Carbohydrates: 0.2 mg/ml.) D-amino acid percentage: 28%D-Ala, 9% D-Ser, 36% D-Asx, 33% D-Met, 32% D-Phe, 31% D-Glx. NOxproduction in mg of active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL(C2), and 2.0 mg/mL (C3): C1: 5.8 μM, C2: 12.1 μM, C3: 6.8 μM.

StSa 046 Example 2.18

Biomass according to Example 1 was diluted to 68 g/L. Alkalinization at0.25 M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 90.72 mg/ml; Prot: 47.68 mg/ml; A.A:9.36 mg/ml; Carbohydrates: 2.48 mg/ml.)

StSa 047 Example 2.19

Biomass according to Example 1 is diluted to 68 g/L. Alkalinization at0.25 M NaOH is performed. The lysis is incubated for 8 days at 35-40° C.under continuous stirring.

StSa 047 Example 2.20

Biomass according to Example 1 is diluted to 60 g/L. Alkalinization at0.33 M NaOH is performed. The lysis is incubated for 2 days at 35-40° C.under continuous stirring.

StSa 048 Example 2.21

Biomass according to Example 1 is diluted to 62 g/L. Alkalinization at0.33 M NaOH is performed. The lysis is incubated for 8 days at 35-40° C.under continuous stirring.

StPy 8191 Example 2.22

Biomass according to Example 1.5 is diluted to 55 g/L. Alkalinization at0.33 M NaOH is performed. The lysis is incubated for 4 days at 35-40° C.under continuous stirring.

StPn 7978 Example 2.23

Biomass according to Example 1.5 was diluted to 38.7 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 66.4 mg/ml; Prot: 25.4 mg/ml;A.A: 6.0 mg/ml; Carbohydrates: 2.5 mg/ml.) NOx production in mg ofactive dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL(C3): C1: 10.2 μM, C2: 20.7 μM, C3: 20.8 μM.

StPn 7978 Example 2.24

Biomass according to Example 1.5 was diluted to 30 g/L. Alkalinizationat 0.022 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 30.4 mg/ml; Prot: 2.20 mg/ml;Carbohydrates: 0.40 mg/ml.)

StPn 7978 Example 2.25

Biomass according to Example 1.5 was diluted to 60 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 59 mg/ml; Prot: 17 mg/ml;A.A: 7.0 mg/ml; Carbohydrates 1.8 mg/ml.) NOx production in mg of activedry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1:9.1 μM, C2: 13.4 μM, C3: 1.4 μM.

StPn 7978 Example 2.26

Biomass according to Example 1.5 was diluted to 12.5 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 2 days at 35-40°C. under continuous stirring. (SDW: 17.8 mg/ml; Prot: <0.2 mg/ml; A.A:2.0 mg/ml; Carbohydrates: 0.7 mg/ml.)

StPn 7978 Example 2.27

Biomass according to Example 1.5 was diluted to 60 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 43.6 mg/ml; Prot: 6 mg/ml; A.A: 10mg/ml; Carbohydrates: 1.5 mg/ml.)

StPn 7978 Example 2.28

Biomass according to Example 1.5 was diluted to 12.5 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 55.4 mg/ml; Prot: 2 mg/ml;A.A: 4 mg/ml; Carbohydrates 0.7 mg/ml.)

StPn 7978 Example 2.29

Biomass according to Example 1.5 was diluted to 60 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 118.4 mg/ml; Prot: 31 mg/ml;A.A: 19 mg/ml; Carbohydrates: 4.1 mg/ml.) NOx production in mg of activedry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1:5.8 μM, C2: 12.1 μM, C3: 2.8 μM.

StPn 7978 Example 2.30

Biomass according to Example 1.5 was diluted to 12.5 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 18.4 mg/ml; Prot: <0.2 mg/ml;A.A: 2 mg/ml; Carbohydrates: 0.5 mg/ml.)

StPn 7465 Example 2.31

Biomass according to Example 1.5 was diluted to 52.5 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 69.4 mg/ml; Prot: 32.3 mg/ml;A.A: 6.0 mg/ml; Carbohydrates: 1.7 mg/ml.)

StPn 7466 Example 2.32

Biomass according to Example 1.5 was diluted to 40.0 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 60.6 mg/ml; Prot: 29.0 mg/ml;A.A: 6.0 mg/ml; Carbohydrates: 1.50 mg/ml.)

StPn 10319 Example 2.33

Biomass according to Example 1.5 was diluted to 41.2 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 60.4 mg/ml; Prot: 28.4 mg/ml;A.A: 6.0 mg/ml; Carbohydrates 1.1 mg/ml.)

StSa 046 Example 2.34

Biomass according to Example 1.5 was diluted to 58.9 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 83.2 mg/ml; Prot: 7.2 mg/ml;A.A: 8.39 mg/ml; Carbohydrates: 2.16 mg/ml.)

StSa 047 Example 2.35

Biomass according to Example 1.5 was diluted to 50.4 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 60.64 mg/ml; Prot: 27.92mg/ml; A.A: 7.2 mg/ml; Carbohydrates: 3.52 mg/ml.)

StSa 048 Example 2.36

Biomass according to Example 1.5 was diluted to 66.2 g/L. Alkalinizationat 0.25 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 72.96 mg/ml; Prot: 36.16mg/ml; A.A: 7.2 mg/ml; Carbohydrates: 3.28 mg/ml.)

NeCa 1045 Example 2.37

One aliquot of NeCa 1045 from Example 1.4 containing 223 g of bacterialmaterial was thawed at room temperature and diluted with a salinesolution (8 g/L NaCl) to reach 22.8 g/L. Alkalinization at 0.2 M NaOHwas performed. The lysis was incubated for 192 hours at 35-40° C. undercontinuous stirring. During the lysis, the pH was monitored so as not todecrease by more than 0.5 pH units. (SDW: 41.29 mg/m; Prot: 17.45 mg/mL;A.A.: 3.41 mg/mL; Carbohydrates: 3.4 mg/mL.) NOx production in mg ofactive dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL(C3): C1: 4.2 μM, C2: 14.8 μM, C3: 13.4 μM.

NeCa 1045 Example 2.38

Biomass according to Example 1.4 was diluted to 20.5 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 3 days at35-40° C. under continuous stirring. (SDW: 36.6 mg/ml; Prot: 16.4 mg/ml;A.A: 3.63 mg/ml; Carbohydrates: 0.77 mg/ml.)

NeCa 1045 Example 2.39

Biomass according to Example 1.4 was diluted to 51.0 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 75.69 mg/ml; Prot: 30.8mg/ml; A.A: 10.8 mg/ml; Carbohydrates: 1.14 mg/ml.) D-amino acidpercentage: 40% D-Ala, 45% D-Asx, 42% D-Met, 40% D-Phe, 46% D-Glx, 48%D-Lys. NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1),0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 4.9 μM, C2: 14.3 μM, C3: 3.6 μM.

NeCa 1045 Example 2.40

Biomass according to Example 1.4 was diluted to 13 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 2 days at 35-40°C. under continuous stirring. (SDW: 15.08 mg/ml; Prot: 7.7 mg/ml; A.A:1.5 mg/ml; Carbohydrates: 0.28 mg/ml.) D-amino acid percentage: 21%D-Ala, 67% D-Ser, 31% D-Asx, 25% D-Met, 12% D-Lys.

NeCa 1045 Example 2.41

Biomass according to Example 1.4 was diluted to 51.0 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 41.23 mg/ml; Prot: 26.2 mg/ml; A.A:4.6 mg/ml; Carbohydrates: 0.98 mg/ml.)

NeCa 1045 Example 2.42

Biomass according to Example 1.4 was diluted to 13 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 48.43 mg/ml; Prot: 7.7 mg/ml;A.A: 4.3 mg/ml; Carbohydrates: 0.22 mg/ml.)

NeCa 1045 Example 2.43

Biomass according to Example 1.4 was diluted to 51.0 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 83.02 mg/ml; Prot: 28.9mg/ml; A.A: 15 mg/ml; Carbohydrates: 1.11 mg/ml.) D-amino acidpercentage: 44% D-Ala, 48% D-Ser, 47% D-Asx, 45% D-Met, 43% D-Phe, 50%D-Glx. NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1),0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 5.1 μM, C2: 12.0 μM, C3: 7.5 μM.

NeCa 1045 Example 2.44

Biomass according to Example 1.4 was diluted to 13 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 15.26 mg/ml; Prot: 8.6 mg/ml;A.A: 1.8 mg/ml; Carbohydrates 0.22 mg/ml.) D-amino acid percentage: 31%D-Ala, 42% D-Ser, 38% D-Asx, 36% D-Met, 35% D-Phe, 37% D-Glx. NOxproduction in mg of active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL(C2), and 2.0 mg/mL (C3): C1: 4.1 μM, C2: 13.9 μM, C3: 6.0 μM.

NeCa 1045 Example 2.45

Biomass according to Example 1.4 was diluted to 9 g/L. Alkalinization at0.066 M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 20.62 mg/ml; Prot: 5.57 mg/ml;Carbohydrates: 0.06 mg/ml.)

NeCa NCTC3622 Example 2.46

Biomass according to Example 1.4 was diluted to 20.1 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 35.69 mg/ml; Prot: 14.25mg/ml; A.A: 3.4 mg/ml; Carbohydrates: 0.71 mg/ml.)

NeCa NCTC3625 Example 2.47

Biomass according to Example 1.4 was diluted to 9.7 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 38.83 mg/ml; Prot: 15.54mg/ml; A.A: 3.4 mg/ml; Carbohydrates: 0.95 mg/ml.)

KlPn 204 Example 2.48

One aliquot of KlPn 204 Example 1.3 containing 440 g of bacterialmaterial was thawed at room temperature and diluted with a salinesolution (8 g/L NaCl) to reach 37.7 g/L. Alkalinization at 0.2 M NaOHwas performed. The lysis was incubated for 192 hours at 35-40° C. undercontinuous stirring. During the lysis, the pH was monitored so as not todecrease by more than 0.5 pH units. (SDW: 51.77 mg/mL; Prot: 27.66mg/mL; A.A.: 4.2 mg/mL; Carbohydrates: 1.03 mg/mL). NOx production in mgof active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL(C3): C1: 0.6 μM, C2: 2.8 μM, C3: 1.8 μM.

KlPn 204 Example 2.49

Biomass according to Example 1.3 was diluted to 9 g/L. Alkalinization at0.013 N NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 25.71 mg/ml; Prot: 4.91 mg/ml;Carbohydrates 0.51 mg/ml.)

KlPn 204 Example 2.50

Biomass according to Example 1.3 was diluted to 99 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 2 days at 35-40°C. under continuous stirring. (SDW: 50.63 mg/ml; Prot: 15.4 mg/ml; A.A:2.9 mg/ml; Carbohydrates: 2.1 mg/ml.) D-amino acid percentage: 5% D-Ala,6% D-Asx.

KlPn 204 Example 2.51

Biomass according to Example 1.3 was diluted to 13 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 52.69 mg/ml; Prot: 5.7 mg/ml;A.A: 2.3 mg/ml; Carbohydrates: 0.3 mg/ml.) D-amino acid percentage: 36%D-Ala, 8% D-Ser, 45% D-Asx, 7% D-Met, 27% D-Phe, 40% D-Glx, 29% D-Lys.NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL(C2), and 2.0 mg/mL (C3): C1: 0.5 μM, C2: 0.8 μM, C3: 6.0 μM.

KlPn 204 Example 2.52

Biomass according to Example 1.3 was diluted to 99 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 136.34 mg/ml; Prot: 58.9mg/ml; A.A: 20.6 mg/ml; Carbohydrates: 3.4 mg/ml.)

KlPn 204 Example 2.53

Biomass according to Example 1.3 was diluted to 13 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 18.06 mg/ml; Prot: 6.3 mg/ml; A.A:1.1 mg/ml; Carbohydrates: 0.3 mg/ml.) NOx production in mg of active dryweight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 0.5μM, C2: 1.6 μM, C3: 1.9 μM.

KlPn 204 Example 2.54

Biomass according to Example 1.3 was diluted to 99 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 58.06 mg/ml; Prot: 20 mg/ml;A.A: 4.6 mg/ml; Carbohydrates 2.7 mg/ml.)

KlPn 204 Example 2.55

Biomass according to Example 1.3 was diluted to 13 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 52.57 mg/ml; Prot: 5.7 mg/ml;A.A: 4.0 mg/ml; Carbohydrates: 0.3 mg/ml.) D-amino acid percentage: 43%D-Ala, 25% Val, 5% D-Ser, 46% D-Asx, 46% D-Met, 45% D-Phe, 44% D-Glx,38% D-Lys. NOx production in mg of active dry weight/mL, 0.02 mg/mL(C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 0.5 μM, C2: 0.6 μM, C3:2.6 μM.

KlPn 5056 Example 2.56

Biomass according to Example 1.3 was diluted to 39.4 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 52.69 mg/ml; Prot: 27.83mg/ml; A.A: 4.0 mg/ml; Carbohydrates: 1.09 mg/ml.)

KlPn 5050 Example 2.57

Biomass according to Example 1.3 was diluted to 34.2 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 48.23 mg/ml; Prot: 26.57mg/ml; A.A: 4.0 mg/ml; Carbohydrates: 1.03 mg/ml.)

StAu I051 Example 2.58

One aliquot of StAu I051 from Example 1.2 containing 375 g of bacterialmaterial was thawed at room temperature and diluted with a salinesolution (8 g/L NaCl) to reach 55.2 g/L. Alkalinization at 0.2 M NaOHwas performed. The lysis was incubated for 192 hours at 35-40° C. undercontinuous stirring. During the lysis, the pH should was monitored so asnot to decrease by more than 0.5 pH units. (SDW: 66.4 mg/mL; Prot: 34.08mg/mL; A.A.: 6.4 mg/mL; Carbohydrates: 0.64 mg/mL.) NOx production in mgof active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL(C3): C1: 0.6 μM, C2: 0.8 μM, C3: 1.4 μM.

StAu I051 Example 2.59

Biomass according to Example 1.2 was diluted to 51 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 9 days at35-40° C. under continuous stirring. (SDW: 65.31 mg/ml; Prot: 25.56mg/ml; A.A: 6.57 mg/ml; Carbohydrates: 0.98 mg/ml.)

StAu I051 Example 2.60

Biomass according to Example 1.2 was diluted to 81 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 2 days at35-40° C. under continuous stirring. (SDW: 137.2 mg/ml; Prot: 51.2mg/ml; A.A: 20.8 mg/ml; Carbohydrates: 1.6 mg/ml.) D-amino acidpercentage: 51% D-Ala, 11% D-Ser, 43% D-Asx, 37% D-Met, 35% D-Phe, 43%D-Glx. NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1),0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 0.6 μM, C2: 0.8 μM, C3: 1.3 μM.

StAu I051 Example 2.61

Biomass according to Example 1.2 was diluted to 13 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 55.7 mg/ml; Prot: 4.8 mg/ml;A.A: 4.8 mg/ml; Carbohydrates: 0.2 mg/ml.)

StAu I051 Example 2.62

Biomass according to Example 1.2 was diluted to 81 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 130.1 mg/ml; Prot: 47.2mg/ml; A.A: 24.8 mg/ml; Carbohydrates: 1.4 mg/ml.)

StAu I051 Example 2.63

Biomass according to Example 1.2 was diluted to 13 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 8 days at 35-40°C. under continuous stirring. (SDW: 18.4 mg/ml; Prot: 4.8 mg/ml; A.A:2.4 mg/ml; Carbohydrates: 0.2 mg/ml.) NOx production in mg of active dryweight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3): C1: 0.5μM, C2: 0.6 μM, C3: 1.3 μM.

StAu I051 Example 2.64

Biomass according to Example 1.2 was diluted to 81 g/L. Alkalinizationat 0.7 M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 134.6 mg/ml; Prot: 48 mg/ml;A.A: 25.6 mg/ml; Carbohydrates: 1.5 mg/ml.) D-amino acid percentage: 56%D-Ala, 10% D-Ser, 45% D-Asx, 42% D-Met, 39% D-Phe, 73% D-Tyr, 49% D-Glx.NOx production in mg of active dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL(C2), and 2.0 mg/mL (C3): C1: 0.6 μM, C2: 0.7 μM, C3: 1.1 μM.

StAu I051 Example 2.65

Biomass according to Example 1.2 was diluted to 13 g/L. Alkalinizationat 0.1M NaOH was performed. The lysis was incubated for 10 days at35-40° C. under continuous stirring. (SDW: 17.9 mg/ml; Prot: 5.6 mg/ml;A.A: 2.4 mg/ml; Carbohydrates: 0.2 mg/ml.) NOx production in mg ofactive dry weight/mL, 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL(C3): C1: 0.5 μM, C2: 0.6 μM, C3: 3.7 μM.

StAu I049 Example 2.66

Biomass according to Example 1.2 was diluted to 52.8 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 70.08 mg/ml; Prot: 34.4mg/ml; A.A: 6.4 mg/ml; Carbohydrates: 0.64 mg/ml.)

StAu I050 Example 2.67

Biomass according to Example 1.2 was diluted to 47.5 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 64.32 mg/ml; Prot: 32.24mg/ml; A.A: 6.4 mg/ml; Carbohydrates: 0.64 mg/ml.)

StAu I050 Example 2.68

Biomass according to Example 1.2 was diluted to 48.5 g/L. Alkalinizationat 0.033 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring.

StAu I052 Example 2.69

Biomass according to Example 1.2 was diluted to 56.9 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 62.72 mg/ml; Prot: 30.8mg/ml; A.A: 6.4 mg/ml; Carbohydrates: 0.72 mg/ml.)

StAu I053 Example 2.70

Biomass according to Example 1.2 was diluted to 67.3 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 68.8 mg/ml; Prot: 32.24mg/ml; A.A: 6.4 mg/ml; Carbohydrates: 1.2 mg/ml.)

StAu I050 Example 2.71

Biomass according to Example 1.2 was diluted to 63.6 g/L. Alkalinizationat 0.2 M NaOH was performed. The lysis was incubated for 8 days at35-40° C. under continuous stirring. (SDW: 68.96 mg/ml; Prot: 24.56mg/ml; A.A: 6.4 mg/ml; Carbohydrates: 2.56 mg/ml.)

Example 3 Purification of Lysates Example 3.1 Clarified Extract of aGram Negative and a Gram Positive Strain

2 L of Example 2.57 and 2 L of Example 2.66 were mixed together, pH wasadjusted to 12.0 with addition of concentrated HCl, and the mixture wasdiluted with 2 L of 8 g/L NaCl solution. The diluted mixture wastransferred to a microfiltration (MF) tank. The microfiltration unitused a 0.45 micron tangential flow filtration filter (PALL Procette PES0.45 micron) in a serpentine mode (See FIG. 1). The cross flow wasadjusted at 2000 L/h m² (LHM) and the trans-membrane pressure (TMP) at1.3 bar. The microfiltration permeate was transferred to anultrafiltration (UF) tank.

Once the volume of the mixture in the microfiltration tank reached thehalf of the initial volume, the UF unit was started. The ultrafiltrationunit used a 30 kDa tangential flow filtration filter (PALL CentrasettePES 30 kD). The crossflow was adjusted at 1000 LHM and the TMP at 0.5bar.

The volumes in the MF and UF tanks were maintained at the same level. Ateach diafiltration volume, the protein concentration was measured by theBradford method. [The Bradford method is standard in the art. There isno need to cite a reference unless different ways of doing the methodgive significantly different results.] In the UF Tank the Bradfordprotein content was 26.8 mg/mL after 1 diafiltration volume (DFV), 34.8mg/mL after 4 DFV, and 37.2 mg/mL after 9 DFV. The permeate flux ofmicrofiltration during the diafiltration was 15 LHM. After 14diafiltration volumes, the UF was stopped, and the product wasconcentrated in the MF tank. The product contained 15.9 mg/mL protein.The product was then diluted to 7.4 mg/mL and filtered through a 0.2micron sterile filter. (Solubilized dry residue (SDR): 21.0 mg/mL, Prot:7.4 mg/mL, A.A.: 1.2 mg/mL, glucides (Carbohydrates): 0.3 mg/mL,Chloride: 4.6 mg/ml.) NOx production in mg of Lowry protein mg/mL, 0.03mg/mL (C1), 0.3 mg/mL (C2), and 3.0 mg/mL (C3): C1: 5.8 μM, C2: 11.4 μM,C3: 1.2 μM.

Example 3.2 Klba Mono Strain

4 kg of Example 2.57 was adjusted to pH 12.0 and diluted with 4 L of 8g/L NaCl solution. The diluted lysate was transferred to amicrofiltration tank.

Microfiltration parameters were: Cross Flow 2000 LHM, TMP 1.3 bar, cutoff: 0.45 μm. Ultrafiltration parameters were: Cross Flow 1000 LHM, TMP0.5 bar, cut off: 30 kDa, number of diafiltration volumes: 8.

The product was diluted to 7.0 mg/mL and filtered through a 0.2 micronsterile filter. (SDR: 18.0 mg/mL, Prot: 7.0 mg/mL, A.A.: 0.8 mg/mL,Carbohydrates: 0.3 mg/mL.) NOx production in mg of Lowry protein/mL,0.03 mg/mL (C1), 0.3 mg/mL (C2), and 3.0 mg/mL (C3): C1: 5.2 μM, C2: 9.8μM, C3: 1.1 μM.

Example 3.3 Moraxella Mono Strain

2 kg of Example 2.37 was adjusted to pH 10.7 and diluted with 3 L of 8g/L NaCl solution. The diluted lysate was transferred to themicrofiltration tank.

Microfiltration parameters were: Cross Flow 2000 LHM, TMP 1.3 bar, cutoff: 0.45 μm. Ultrafiltration parameters were: Cross Flow 1000 LHM, TMP0.5 bar, cut off: 30 kDa, number of diafiltration volumes: 8.

The product was diluted to 7.0 mg/mL and filtered through a 0.2 micronsterile filter. (SDR: 19.4 mg/mL, Prot: 6.8 mg/mL.)

Example 3.4 Pneumoniae Mono Strain

5.0 kg of Example 2.32 was adjusted to pH 12.27 and diluted with 5.0Liters of 8 g/L NaCl solution. The diluted lysate was transferred to amicrofiltration tank.

Microfiltration parameters were: Cross Flow 2000 LHM, TMP 1.3 bar, cutoff: 0.45 μm. Ultrafiltration parameters were: Cross Flow 1000 LHM, TMP0.5 bar, cut off: 30 kDa, number of diafiltration volumes: 8.

The product was diluted to 7.0 mg/mL and filtered through a 0.2 micronsterile filter. (SDR: 23.3 mg/mL, Prot: 4.3 mg/ml.)

Example 3.5

500 ml of Example 2.45, 500 ml of Example 2.47, and 500 ml of Example2.37 are mixed and centrifuged at 9000×g. The supernatant is filteredthrough successive filters with porosities of 0.8 μm, 0.45 μm and 0.2μm. The pH is adjusted to 10.5 with HCl.

Example 3.6

300 ml of the lysate of Example 2.23, 300 ml of Example 2.31, 300 ml ofExample 2.32, and 300 ml of Example 2.33 were mixed and centrifuged for30 minutes at 9000×g. The supernatant was filtered through successivefilters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH wasadjusted to 10.5 with HCl. Analytical results: (SDR: 63.60 mg/mL, Prot:23.00 mg/mL, A.A.: 6.00 mg/mL, Carbohydrates: 1.60 mg/ml.)

Example 3.7

300 ml of Example 2.48, 300 ml of Example 2.56, and 300 ml of Example2.57 were mixed and centrifuged at 9000×g. The supernatant was filteredthrough successive filters with porosities of 0.8 μm, 0.45 μm and 0.2μm. The pH was adjusted to 10.5 with HCl. (SDR: 50.40 mg/mL, Prot: 22.30mg/mL, A.A.: 4.0 mg/mL, Carbohydrates: 0.97 mg/ml.) NOx production in mgof solubilized dry weight/mL, 0.01 mg/mL (C1), 0.1 mg/mL (C2), and 1.0mg/mL (C3): C1: 1.06 μM, C2: 3.19 μM, C3: 7.62 μM.

Example 3.8

200 ml of Example 2.58, 200 ml of Example 2.66, 200 ml of Example 2.67,200 ml of Example 2.69, 200 ml of Example 2.70 and 200 ml of Example2.71 were mixed and centrifuged for at 9000×g. The supernatant wasfiltered through successive filters with porosities of 0.8 μm, 0.45 μmand 0.2 μm. The pH was adjusted to 10.5 with HCl. (SDR: 65.12 mg/mL,Prot: 24.80 mg/mL, A.A.: 7.2 mg/mL, Carbohydrates: 1.12 mg/ml.) NOxproduction in mg of solubilized dry weight/mL, 0.01 mg/mL (C1), 0.1mg/mL (C2), and 1.0 mg/mL (C3): C1: 0.76 μM, C2: 1.16 μM, C3: 3.79 μM.

Example 3.9

500 ml of Example 2.46, 500 ml of Example 2.47 and 500 ml of Example2.37 were mixed and centrifuged for at 9000×g. The supernatant wasfiltered through successive filters with porosities of 0.8 μm, 0.45 μmand 0.2 μm. The pH was adjusted to 10.5 with HCl. (SDR: 38.15 mg/mL,Prot: 13.5 mg/mL, A.A.: 3.4 mg/mL, Carbohydrates: 0.80 mg/ml.) NOxproduction in mg of solubilized dry weight/mL, 0.01 mg/mL (C1), 0.1mg/mL (C2), and 1.0 mg/mL (C3): C1: 2.25 μM, C2: 5.25 μM, C3: 14.21 μM.

Example 3.10

500 ml of Example 2.34, 500 ml of Example 2.35, 500 ml of Example 2.36,and 500 ml of Example 2.10 were mixed and centrifuged at 9000×g. Thesupernatant was filtered through successive filters with porosities of0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCl. (SDR:69.6 mg/mL, Prot: 28.00 mg/mL, A.A.: 7.2 mg/mL, Carbohydrates: 2.48mg/ml.) NOx production in mg of solubilized dry weight/mL, 0.01 mg/mL(C1), 0.1 mg/mL (C2), and 1.0 mg/mL (C3): C1: 0.95 μM, C2: 1.21 μM, C3:4.44 μM.

Example 3.11

10 ml of the lysate of Example 2.61 and 10 ml of Example 2.27 werecentrifuged at 9000×g, separately. The supernatants were filteredthrough successive filters with porosities of 0.8 μm, 0.45 μm and 0.2μm. 3 mL of each supernatant were mixed. The pH was adjusted to 7.2 withHCl. NOx production in mg of solubilized dry weight/mL, 0.01 mg/mL (C1),0.1 mg/mL (C2), and 1.0 mg/mL (C3): C1: 0.34 μM, C2: 0.66 μM, C3: 1.33μM.

Example 3.12

10 ml of Example 2.58 and 10 ml of Example 2.61 were centrifuged at9000×g, separately. The supernatants were filtered through successivefilters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. 3 mL of eachsupernatant were mixed together. The pH was adjusted to 7.6 with HCl.NOx production in mg of solubilized dry weight/mL, 0.01 mg/mL (C1), 0.1mg/mL (C2), and 1.0 mg/mL (C3): C1: 0.40 μM, C2: 0.44 μM, C3: 0.71 μM.

Example 3.13

10 ml of Example 2.9 and 10 ml of Example 2.8 were centrifuged at9000×g, separately. The supernatants were filtered through successivefilters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. 1.5 mL of thefirst lysate (Example 2.9) was mixed with 4.5 mL of the second (Example2.8). The pH was adjusted to 7.2 with HCl. NOx production in mg ofsolubilized dry weight/mL, 0.01 mg/mL (C1), 0.1 mg/mL (C2), and 1.0mg/mL (C3): C1: 8.7 μM, C2: 16.90 μM, C3: 21.1 μM.

Example 3.14

10 ml of Example 2.2 and 10 ml of Example 2.8 were centrifuged at9000×g, separately. The supernatant was filtered through successivefilters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. 3 mL of eachsupernatant were mixed together. The pH was adjusted to 7.5 with HCl.NOx production in mg of solubilized dry weight/mL, 0.01 mg/mL (C1), 0.1mg/mL (C2), and 1.0 mg/mL (C3): C1: 9.2 μM, C2: 11.3 μM, C3: 17.2 μM.

Example 3.15

13.2 L of lysis from Example 2.2, 48.6 L of mixture lysis from Example3.9, 36.6 L of mixture lysis from Example 3.7, 36 L of mixture lysisfrom Example 3.8, 14.4 L of Example 3.6, and 18.4 L of Example 3.10 weremixed together and diluted to 334.4 L with a 8 g/L NaCl solution. Thesolution was purified by tangential flow filtration in a doublemicrofiltration and ultrafiltration loops system, as described inExample 3.1. (SDW: 21.0 mg/ml; Prot: 6.4 mg/ml; A.A: 1.9 mg/ml;Carbohydrates 0.45 mg/ml.) D-amino acid percentage: 42% D-Ala, 12% Leu,53% D-Ser, 37% D-Asx, 35% D-Met, 32% D-Phe, 28% D-Glx, 8% D-Tyr, 16%Lys. NOx production in mg of Lowry protein mg/mL, 0.03 mg/mL (C1), 0.3mg/mL (C2), and 3.0 mg/mL (C3): C1: 9.3 μM, C2: 14.3 μM, C3: 11.5 μM.

Example 4 Comparative Example of Bacterial Lysis in Lower pH Conditions

Example 4 represents a process which was performed in alkalineconditions outside the scope of the invention. NaOH concentrations usedto perform the lysis of bacteria were lower than 0.1%, leading to lowerpH values.

The following lysates were mixed: 1.18 kg of Example 2.24, 3.0 kg ofExample 2.49, 2.95 kg of Example 2.69, 1.08 Kg of Example 2.4, 1.51 kgof Example 2.11, and 3.98 kg of Example 2.45. Six kilograms of themixture was diluted to 12 kg with NaCl Solution 8 g/L and the pH wasadjusted to pH 12.0. Microfiltration parameters were: Cross Flow 2000LHM, TMP 1.3 bar, cut off: 0.45 μm. Ultrafiltration parameters were:Cross Flow 1000 LHM, TMP 0.5 bar, cut off: 30 kDa, number ofdiafiltration volumes: 12. (SDR: 19.4 mg/ml, Prot: 3.1 mg/ml, A.A.: 2.0mg/ml, Carbohydrates: 0.1 mg/ml, LAL: 20140 EU/ml.) NOx production in mgof active dry weight/mL, 0.03 mg/mL (C1), 0.3 mg/mL (C2), and 3.0 mg/mL(C3): C1: 9.3 μM C2: 14.3 μM C3: 11.5 μM.

Example 5 Lysis of Lactobaccilus Helveticus

Aliquots of bacterial biomass of Lactobacillus helveticus obtained byfermentation on a vegetal medium are thawed at room temperature anddiluted with purified water to reach 80 g/L of dry weight concentration.Alkalinization at 0.2 M NaOH is performed. The lysis is incubated for 2days at 35-40° C. under continuous stirring. During the lysis, the pH ismonitored so as not to decrease by more than 0.5 pH units.

Example 6 Immunoprotection against Aerosol Influenza Virus Infection inMice

This experiment was aimed at investigating the non-specificimmunological activity of certain embodiments of the invention byevaluating their efficacy against a virus, namely H1N1. Six-week-oldfemale BALB/c mice were purchased from Bundesinstitut fürRisikobewertung, Berlin, and were used for all experiments. The animalswere maintained under normal conditions at ambient temperature of 22° C.and a relative humidity of 60±5%. The light program was set on alight-dark cycle of 12:12 hours. Animals were fed with a standard dietof pellets (Altromin 1314, Altromin, Lage, Germany) and tap water wasprovided ad libitum.

Pre-Treatment

A total of 60 mice were divided into 3 groups of 20 mice each, with 2treatment groups and 1 control group. Animals (2 groups) werepre-treated with either 1 mg or 10 mg per mouse once daily for 10consecutive days, or PBS control (1 group). At the end of thepre-treatment, all mice were given an aerosol of a mouse-adaptedA/PR/8/34 (H1N1) influenza virus. The LD₅₀ for mice using this strain isusually 10^(−4.5) by the intranasal and 10^(−1.7) by the aerosol route.The dosage used for challenge infection in these experiments wasselected to provide a dilution capable of causing clear symptomaticinfluenza virus infection, but not 100% lethality.

Mortality was observed was daily in groups of 10 mice for 10 days. Theresults presented in FIG. 3 show that a 10 mg/mouse dose of the extractunder the dosing conditions of this experiment could confer completeprotection against re-infection. In contrast, only 70% of the controlmice survived the influenza virus infection. Survival in the groupreceiving 1 mg/mouse of the drug was 70%.

Clinical Symptoms Observed in Treated Mice after Influenza VirusInfection

Groups of 10 mice each were observed daily for clinical symptoms ofinfluenza virus infection. The clinical symptoms score is shown in thetable below. Animals administered 10 mg dose of the extract showed mildclinical symptoms 2 days later and were apparently healthy earlier thanthe control group mice, indicating beneficial effect of higher dose.There were no significant differences in clinical scores between 1 mgdose-treated groups and controls.

Days after Influenza virus infection Group 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 PBS 0 0 + + ++ +++ +++ +++ +++ +++ +++ ++/ ++/ ++ ++ ++ +++ +++TTT 0 0 + + ++ ++ +++ +++ +++ +++ +++ +++ ++/ ++ ++ +  1 mg +++ TTT 0 00 0 + +/ ++ +++ +++ ++/ ++/ ++/ +/ ++ ++ 0 10 mg ++ +++ +++ +++ +++ TTT= Treatment with Example 16 lysate Only surviving animals were takeninto consideration for the clinical symptoms score. Code: 0: no rufflingof the fur, appeared healthy, and + to +++: progressive increase inruffling of the fur; progressive decrease in movement and generalactivity.

Influenza Hemgglutination Inhibition Antibody Titers of Treated Mice

Sera were collected on day 10 after influenza virus challenges, 3 miceper group, after the first and second infections. Sera obtained fromimmunized animals were stored at −20° C. All sera were pretreated withreceptor-destroying enzyme to remove nonspecific inhibitors. Influenzahemagglutination-inhibition (HI) tests were performed in microtiterplates using 0.5% chicken red blood cells and 4 hemagglutinating unitsusing standard procedures recommended by the World Health Organization(WHO, 2002). The results showing geometric mean HI antibody titersagainst influenza A/PR/8/34 (H1N1) virus are presented in the tablebelow. There were no significant differences between the treated groupsand controls after the first infection.

Group Titer D13 * GMT Titer D34 * GMT PBS 320 403 640 640 320 640 640640 TTT 320 403 320 320  1 mg 320 320 640 320 TTT 320 320 640 1612 10 mg320 2560 320 2560 TTT = Treatment with Example 16 Sera were obtained 10days after influenza virus challenge infection. * Geometric mean titre

Therefore, mice were given second (booster) challenge infection withA/PR/8/34 influenza virus 21 days after the first (primary) infection.The magnitude of antibody titers increased after challenge withA/PR/8/34 (H1N1) virus as shown above. Higher levels of HI antibodytiters were observed with 10 mg/mouse dose of OM-85 BV. In contrast, 1mg/mouse dose had lower antibody titers than 10 mg/mouse dose of thedrug.

There were no significant differences between treated groups andcontrols in the lung virus detected. Nonetheless, clinical symptomsafter aerosol infection with a mouse-pathogenic influenza A/PR/8/34virus in animals administered 10 mg dose of an extract according to theinvention occurred at least one complete day later than controls, andthe experimental group also recovered apparently earlier than controlmice. However, there were no significant differences in clinical scoresbetween 1 mg dose treated groups and controls, suggesting the effect ofthe extract was dose-dependent. Three days after the first infection,there were no significant differences in influenzahemagglutination-inhibition antibody levels between the treated groupsand controls. However, upon second infection with A/PR/8/34 influenzavirus three weeks after the first infection, a higher and significantmagnitude of antibody induction was observed with 10 mg/mouse dosegroup.

This example shows that an extract according to the invention at thedosage of 10 mg per mouse was capable of conferring protection againstmouse-pathogenic aerosol influenza A/PR/8/34 (H1N1) virus infection asdetermined by multiple parameters. Embodiments of the invention maytherefore activate in vivo an immune response against a virus. Since theextracts may be, in some embodiments, manufactured exclusively frompathogenic bacteria, as was the extract tested in this example, thissuggests that embodiments of the invention may activate the innateimmune system.

Example 7 Activity of Embodiments of the Invention in a Murine NitricOxide Test

Six-week old male C57/BL6 mice (six weeks old male, SPF quality, CharlesRivier, FR) were killed by CO₂ inhalation. The hip, femur, and tibiafrom the posterior appendage were removed. The bone marrow was extractedfrom the lumen by injecting Dulbecco's Modified Eagle Medium (DH)through the bone after cutting both end portions. After washing, thestem cells were resuspended (40,000 cells/mL) in DH medium supplementedwith 20% horse serum and 30% L929 cell supernatant. The cell suspensionwas incubated for 8 days in an incubator at 37° C. under 8% CO₂ andmoisture-saturated atmosphere. Macrophages were then detached withice-cold PBS, washed and resuspended in DH medium supplemented with 5%fetal calf serum (FCS), amino acids and antibiotics (DHE medium). Thecell density was adjusted to 700,000 cells/mL. Aqueous solutions of theproducts were serially diluted in DHE medium directly in microtiterplates. The products were tested in triplicates and each microtiterplate comprised a negative control composed of medium. The final volumein each well was 100 μL. 100 μL of the cell suspension was added to thediluted products and the cells were incubated for 22 h in an incubatorat 37° C., under 8% CO2 and a moisture-saturated atmosphere. At the endof the incubation period, 100 μL of supernatant was transferred toanother microtiter plate and the nitrite concentration produced in eachsupernatant was determined by running a Griess reaction. 100 μL ofGriess reagent (5 mg/mL of sulfanilamide+0.5 mg/mL ofN-(1-naphtyl)ethylene-diamine hydrochloride) in 2.5% aqueous phosphoricacid was added to each well. The microtiter plates were read with aspectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm againsta reference at 690 nm. The nitrite concentration was proportional tonitric oxide content being formed. The nitrite content was determinedbased on a standard curve. The results were given in μM nitric oxide(NO) as mean value±standard deviation and plotted as a dose responsecurve (See FIGS. 4-7).

Example 8 Limulus Amoebocyte Lysate Chromogenic (LAL) Test

To determine the presence of endotoxin-like molecules, an LAL test wasperformed with the Chromogenic—LAL Kit of Bio-Whittaker.

This test is based on activation by lipopolysaccharide (LPS) or productsof comparable structure, by an enzymatic cascade present in the LAL.This enzymatic activation is demonstrated by the splitting of achromogen linked to a peptide by a protease. The enzymatic reaction iscarried out at 37° C. and the formation of the chromogen over time ismeasured at 405 nm. The time necessary to reach 0.2 units of D.O. isrecorded and the endotoxic activity calculated in relation to a LPSstandard (standard curve).

The results of such an example experiment on extracts according to theinvention are expressed in the table below in EU (Endotoxin Unit) inrelation to a standardized preparation of E. coli LPS (1 EU correspondsto 0.09 ng equivalent LPS).

Conditions of lysis (Time of lysis, amount of Starting Material, ngequivalent initial percentage of NaOH) EU/ml LPS/ml Hemo NaOH 1% 100g/l/T 22 h 169900 ± 600  15291 Hemo NaOH 1% 125 g/l/T 211 h 2811000 ±82644 252990 Hemo NaOH 1% 50 g/l/T 211 h 33950 ± 224 3055 Hemo NaOH 1%50 g/l/T 22 h >5000000 450000 Hemo NaOH 1% 50 g/l/T 92 h >500000 45000Hemo NaOH 2% 25 g/l/T 92 h 65430 ± 565 5888 Hemo NaOH 2% 50 g/l/T 211 h23560 ± 142 2120 Hemo NaOH 2% 50 g/l/T 22 h 579200 ± 1062 52128 HemoNaOH 2% 50 g/l/T 92 h  7927 ± 320 713 Hemo NaOH 3% 125 g/l/T 211 h 30000± 113 2700 Hemo NaOH 3% 25 g/l/T 22 h 6883 ± 34 619 Hemo NaOH 3% 50g/l/T 22 h 10690 ± 41  962 Hemo NaOH 4% 100 g/l/T 92 h 11380 ± 36  1024Hemo NaOH 4% 12.5 g/l T 22 h 3415 ± 40 307 Hemo NaOH 4% 12.5 g/l T 92 h10500 ± 37  945 Hemo NaOH 10% 100 g/l T 92 h 1229 ± 38 110 Hemo NaOH 10%12.5 g/l/T 211 h  224 ± 17 20 Hemo NaOH 10% 125 g/l/T211 h 602 ± 6 54Example 4 20140 1812 Example 3.15   108 ± 0.5 10 H₂O <0.005 15291

Example 9 Inhibition of Histamine Secretion

An in vitro rat model of mast cell degranulation (proposed by the CROCEREP, catalog number 2006: 771-c, ref Håkanson R, Rönnberg A L, SjölundK. Anal Biochem, 1972 June 47(2):356-70) was employed to investigate theway in which an embodiment of the invention (comprising 21 bacterialstrains) would inhibit histamine secretion by compound 48/80-stimulatedmastocytes. The protocol and experimental conditions are brieflysummarized in the tables below:

Protocol

Reference Assay Origin Compound Reference Histamine secretion rat mastSCG Hakanson et (compound cells al. (1972) 48/80-stimulated)

Experimental Conditions

Reaction Method of Assay Stimulus Incubation Product Detection Histaminesecretion Compound 2 min./ histamine Fluorimetry (compound 48/80 37° C.48/80-stimulated) (0.1 μg/ml)

Analysis and Expression of the Results

The results are expressed as a percent of control specific activity:(measured specific activity/control specific activity)×100, obtained inthe presence of the test compounds. The IC50 values (concentrationcausing a half-maximal inhibition of control specific activity) weredetermined by non-linear regression analysis of the inhibition curvesgenerated with mean replicate values using Hill equation curve fitting(Y=D+[(A−D)/(1+(C/C50)nH)], where Y=specific activity, D=minimumspecific activity, A=maximum specific activity, C=compoundconcentration, C50=IC50, and nH=slope factor). This analysis wasperformed using software developed at Cerep (Hill software) andvalidated by comparison with data generated by the commercial softwareSigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.).

In each experiment, the reference was tested concurrently with the testextract in order to assess the assay suitability. Several concentrationswere tested (for IC₅₀ value determination), and the data were comparedwith historical values determined at Cerep. The assay was consideredvalid if the suitability criteria were met, in accordance with thecorresponding standard operating procedure. The IC₅₀ values determinedfor the test extract and the reference (measured twice) are indicated inthe table below. The IC₅₀ values for the reference were within acceptedlimits of the historic average ±0.5 log units.

The corresponding inhibition curves obtained with the test extract areshown in FIG. 8.

Compounds tested IC₅₀ Test Extract 0.0049 mg/ml SCG (reference 1)4.7E−06M SCG (reference 2) 1.1E−06M

The results indicate that the tested extract is a potential inhibitor ofhistamine secretion induced by compound 48/80-stimulated mast cells.

Example 10 Effect of an Embodiment of the Invention in an Escherichiacoli Infection Model in an LPS-Insensitive Strain of Mice

An embodiment of the invention was tested in a recognized in vivo modelof E. coli bacterial infections (See Hopkins et al., Inheritance ofsusceptibility to induced Escherichia coli bladder and kidney infectionsin female C3H/HeJ mice., J Infect Dis. 2003 Feb. 1; 187(3):418-23.).C3H/HeJ mice were mutated for the toll-like receptor gene (TLR4), andare insensitive to TLR4 agonists such as LPS. Therefore this model issuitable to detect the effects of drugs acting via other routes thanTLR4.

One group of 10-12 weeks old female C3H/HeJ mice (8 mice) was treatedorally with an extract similar to that of Example 3.15 for 10 days priorto E. coli infection.

The animals were maintained under normal conditions at ambienttemperature of 18-26° C. and a relative humidity of 30 to 70%. The lightprogram was set on a light-dark cycle of 12:12 hours.

Animals were fed a standard diet provided by Harlan Sprague Dawley(Indianapolis, Ind.) laboratories. Tap water was provided ad libitum,unless when indicated elsewhere.

Treated animals received orally 143 mg of lyophilizate (i.e. 25 mg(17.5%) of bacterial extract and 118 mg (82.5%) of excipients) peranimal per administration of the extract.

Inoculation

Mice were inoculated intravesically with PBS or with uropathogenic E.coli, according to a minimal inoculum protocol that greatly reduces thelikelihood of reflux-associated inoculation of the kidneys and inducesinfections in all animals inoculated. The E. coli strain 1677, isolatedfrom the urine of a woman with a febrile UTI, was used in theseexperiments. To prepare the inoculum, bacteria were grown from frozenstock by 2 passages in tryptose broth (Difco Laboratories), washed withPBS, and resuspended to a concentration of 2×10¹⁰ bacteria/mL. Mice weredeprived of water for 1 h and had urine expressed from their bladdersimmediately before inoculation. Ten microliters of bacterial inoculumwere instilled into the bladder by urethral catheterization underisoflurane anesthesia, resulting in a dose of 2×10⁸ E. coli per mouse.The animals were allowed to recover from anesthesia and water was givenback 1 h later.

Mice were killed 10 days after inoculation to assess the intensities ofbladder and kidney infections. The bladder and both kidneys of eachanimal were removed, weighed, and homogenized in sterile PBS, afterwhich the homogenates were serially plated onto Levine's eosin-methyleneblue agar (Difco Laboratories). The number of E. coli colonies on eachplate was counted after overnight incubation at 37° C. and was used tocalculate the total number of bacteria in each bladder or pair ofkidneys.

Fisher's protected least significant difference test was used todetermine the statistically significant differences between the meantotal colony-forming unit (CFU) values for different groups of mice(PBS, Untreated infected group, and Treated and infected group). Thebladder and kidney infection data was transformed using total CFU=log 10[(CFU+100)/mg tissue], where CFU was the total number of colony-formingunits calculated per tissue sample.

The results obtained are shown in FIGS. 9 a and 9 b for bladder andkidneys, respectively. Mice were sacrificed 10 days after inoculation toassess the intensities of bladder and kidney infections. The bladder andboth kidneys of each animal were removed, weighed, and homogenized insterile PBS, after which the homogenates were serially plated ontoLevine's eosin-methylene blue agar (Difco Laboratories). The number ofE. coli colonies on each plate was counted after overnight incubation at37° C., and after the addition of 100 CFU to the results obtained, thesum obtained in each case was divided by the mg of tissues (i.e. totalCFU=log 10 [(CFU+100)/mg tissue] in order to calculate the mean totalnumber of bacteria +/−SEM for bladders or kidneys. The bacterial extractdecreased by a factor >3 (bladder) and >2 (kidneys) the logarithmicvalues obtained, suggesting that the number of colonies cultured fromthe bladder and the kidneys was decreased by at least a factor of 1000and 100, respectively. These results demonstrate the immunologicalactivity of an embodiment of the invention.

Example 11 Effect of the an Embodiment of the Invention in a MurineModel of Intraperitoneal Salmonella typhimurium Infection in C57/bl Mice

An embodiment of the invention was also tested in a murine model ofintraperitoneal Salmonella typhimurim infection. C57BL/6 mice were keptfor 7 days before oral treatment. Treated animals received 85 mg oflyophilizate (i.e. 15 mg (17.5%) of bacterial extract and 70 mg (82.5%)of excipients) per animal per administration. The experiment consistedof one experimental group of 20 mice treated with an extract similar tothat of Example 3.15, and a control group of 20 mice treated with awater control. For treatment, the extract was dissolved daily indistilled water in order to have a single dose in a final volume of 0.5ml. This 0.5 ml volume was given to each mouse orally once a day for 10consequent days before all mice were challenged intraperitoneally withSalmonella typhimurium strain 415 (I. Mechnokov Institute for Vaccinesand Sera, Russian Academy of Medical Sciences).

The extract was introduced in a single dose of 85 mg per mouse (i.e. 15mg of active principle with 82.5% of excipients). Mice in the controlgroup received a sham treatment using oral administration of 0.5 mlwater daily for 10 days. A preliminary dose-finding challenge rangedfrom 102 to 105 CFU of Salmonellae per mouse. The dose of 104 CFU wasselected the main experiment because this dose provided approximately50% of survivors in untreated animals.

After the challenge, mice were kept under the standard conditions forlaboratory animals. Daily observation and records of death wereperformed during a period of 21 days post-infection. The anti-infectiveefficacy of the extract (see tables below) was estimated according tothe post-infection survival rate (SR), and the post-infection averageduration of life (ADL), and the defense factor (DF), and the preparationefficacy index (EI), which were calculated for each experimental group.The SR was taken as a percent of live animals in the experimental groupon day 21 post-infection.

The ADL, DF and EI were calculated using the following formulas:

ADL=(X1+X2+ . . . +Xn):N,

where ADL is an average duration of life, X1 to Xn are durations of lifepost-infection for experimental mice #1 to #n, and N—is a total numberof animals in the experimental group.

DF=CD:ED,

where DF is the defense factor, CD is a percent of death in the controlgroup, and ED is a percent of death in the experimental group.

EI=[(DF−1):DF]×100%,

where EI is the preparation efficacy index and DF is the defense factor.

Death records in control and experimental group during the period of 21days post-infection with 10⁴ CFU of Salmonella typhimurium.

Nr of mice Pre- before Number of dead mice from day 1 to day 21 postinfection Survival treatment challenge 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 19 20 21 Rate (%) H₂O 19 — — — 1 2 2 — 1 — — — 1 — 1 — — — —— — — 58 Example 19 — — — — — — — — — — — — — — — — — — — — — 100 3.15Defense Efficacy of Example 3.15 in the Model of Salmonella thyphimuriumLethal Infection in C57BL/6 mice.

Pre-Treatment Death Survival ADL With Substances Rate (%) Rate (%)(days) DF EI (%) H₂O 42 58 15.3 1 0 Example 0 100 21 Maximum 100 3.15defense

During the experiment it was evident that the extract appeared welltolerated. In the control group of mice pre-treated with water, thesurvival rate during the period of observation (21d) was 58%, and theADL occurred to be 15.3 days. In contrast, all the mice that receivedthe extract according to this invention survived to the challenge. Theseresults suggest that embodiments of the Invention could be beneficialagainst certain bacterial infections in human beings.

Example 12 Effect of an Embodiment of the Invention on Production ofRegulatory T Cells in Mucosal Trachea During an Allergen Challenge

The immuno-regulatory potential of an embodiment of the invention in amodel of acute allergic inflammation was tested. The experiment wasperformed to determine if, in PVG rats, administration of the an extractaccording to the invention would increase the pool size of availablemucosal-homing T regulatory (Treg) cells, resulting in increased numbersof Tregs in airway mucosal tissues during episodic inflammation.

Inbred PVG rats were bred and maintained free of common rat pathogens.Randomly selected animals of both sexes aged 8-13 weeks were utilizedthroughout.

The daily consecutive feeds (gavage) were performed with 400 ug oflyophilizate provided/g of body (or 400 mg/kg/day). Treg populations inairway tissue of PVG rats were phenotypically identified in the studygroups. Phenotypic characterisation of rat tracheal tissue requiredgroup sizes of 5 to 10 animals pooled to yield sufficient cells for theanalyses. Tracheal tissues were digested in Collagenase/DNase to yieldsingle cell suspensions followed by flow cytometric analysis of surfaceexpression of CD4, CD25, Foxp3, and TCRαβ. First, animals weresensitised with OVA on day zero (d0) and fed with an extract similar tothat of Example 3.15 or placebo from d10 to d17; on d18 they werechallenged with aerosolised OVA and resulting Th cell/Treg (FoxP3)response measured 24 hrs later. For intracellular staining of Fox p3,anti-mouse/rat FoxP3-FLR staining kit from eBioscience (San Diego,Calif.) was used as described by the manufacturer. Data were acquired ona FACSCalibur flow cytometer (BD Biosciences) and analysed using Flowjosoftware (version 4.6.1, Tree Star Inc). OVA was from Sigma ChemicalsCo. (St Louis, Mo.).

FIGS. 10A-10D show original flux cytometry data (in FIGS. 10A and 10B,the markers were CD4 vs FoxP3, and in FIG. 10 b the markers were TCR vsFoxP3). The cells were obtained from airway mucosa after in OVAsentisized rats (i.p. on day 0) and 24 hours after aerosol OVA challenge(day 18). FIGS. 10B and 10D show that when the animals were dosed orallyfrom day 10 to day 18 with the extract, there was an increasedpercentage of FoxP3 positive CD4 cells (and TCR respectively) whencompared to controls shown in FIGS. 10A and 10C (untreated animalssensitized with OVA and OVA challenged animals).

This example shows that embodiments of the invention may havetherapeutic value in case of inflammatory allergic crisis.

Additional Examples Include

An extract from one or more bacterial species chosen from: Moraxellacatarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus sanguinis, Staphylococcus Hemolyticus, Enterococcusfaecalis, Streptococcus mutans, Streptococcus anginosus, Streptococcusmitis, Streptococcus salivarius, Neisseria sicca, Haemophilusparainfluenzae, Actinobacillus (Hemophilus) actinomycetemcomitans, andEikenella corrodens, wherein, during preparation of said extract, theone or more bacterial strains are lysed at a pH of greater than 12, andthe extract is treated so as to remove nucleic acids; and wherein theextract does not pose a risk of prion diseases upon administration to apatient.

The extract of the preceding paragraph obtained from at least one strainof each of the following bacterial species: Moraxella catarrhalis,Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcussanguinis, Staphylococcus Hemolyticus, Enterococcus faecalis,Streptococcus mutans, Streptococcus anginosus, Streptococcus mitis,Streptococcus salivarius, Neisseria sicca, Haemophilus parainfluenzae,Actinobacillus (Hemophilus) actinomycetemcomitans, and Eikenellacorrodens.

The extract of the preceding paragraph obtained from each of thefollowing bacterial strains: Moraxella (Moraxella) catarrhalis 3622,Moraxella (Moraxella) catarrhalis 3625, Moraxella (Moraxella)catarrhalis I-045, Haemophilus influenzae 8467, Klebsiella pneumoniaessp. ozaenae 5050, Klebsiella pneumoniae ssp. pneumoniae 204, Klebsiellapneumoniae ssp. pneumoniae 5056, Staphylococcus aureus I-049,Staphylococcus aureus I-050, Staphylococcus aureus I-051, Staphylococcusaureus I-052, Staphylococcus aureus I-053, Staphylococcus aureus I-054,Streptococcus pneumoniae 7465, Streptococcus pneumoniae 7466,Streptococcus pneumoniae 7978, Streptococcus pneumoniae 10319,Streptococcus pyogenes 8191, Streptococcus sanguinis I-046,Streptococcus sanguinis I-047, Streptococcus sanguinis I-048,Staphylococcus Hemolyticus 11042, Enterococcus faecalis 103015,Streptococcus mutans 10449, Streptococcus anginosus 10713, Streptococcusmitis 12261, Streptococcus salivarius 102503, Neisseria sicca 103345,Haemophilus parainfluenzae 7857, Actinobacillus (Hemophilus)actinomycetemcomitans 52.105, and Eikenella corrodens 10596.

The extract of any of the three preceding paragraphs, wherein theextract comprises less than 100 μg/mL nucleic acid.

The extract of any of the three preceding paragraphs, wherein theextract comprises at least 0.3 mg/mL of saccharides.

The extract of any of the three preceding paragraphs, wherein theextract comprises between 0.3 and 4.5 mg/mL of saccharides.

The extract of any of the preceding paragraphs, wherein at least onesaccharide is selected from the group consisting of monosaccharides,disaccharides, and polysaccharides.

The extract of the preceding paragraph, wherein at least onepolysaccharide is a branched polysaccharide.

The extract of any of the preceding paragraphs, wherein at least onesaccharide is chemically modified.

The extract of any of the preceding paragraphs, wherein the extractcomprises between 1.5 to 2.5 mg/mL of free amino acids.

The extract of any of the paragraphs above, wherein lysis is performedat a pH of 12.6 to 13.4.

The extract of any of the preceding paragraphs, wherein the extract istreated so as to remove particulate and/or insoluble components.

The extract of any of the preceding paragraphs, wherein each bacterialstrain is cultured in a medium that does not pose a risk of priondiseases.

The extract of any of the preceding paragraphs, wherein at least oneamino acid chosen from aspartic acid, glutamic acid, serine, histidine,alanine, arginine, tyrosine, methionine, phenylalanine, and lysine isracemized by at least 10%.

The extract of any of the preceding paragraphs, wherein the free aminoacids of the extract comprise between 1 and 80% D-amino acids.

The extract of any of the preceding paragraphs, wherein the free aminoacids of the extract comprise between 10 and 45% D-amino acids.

The extract of the preceding paragraph, wherein the free amino acids ofthe extract comprise between 25 and 35% D-amino acids.

The extract of any of the preceding paragraphs, wherein the extractcomprises at least one D-amino acid chosen from D-aspartic acid andD-asparagine, D-glutamic acid and D-glutamine, D-serine, D-methionine,D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine,D-leucine, D-lysine, D-valine, and D-threonine.

The extract of the preceding paragraph, wherein the concentration of anyone D-amino acid comprises between 1 and 50% of the free amino acidconcentration.

The extract of the preceding paragraph, wherein the concentration of anyone D-amino acid comprises between 10 and 40% of the free amino acidconcentration.

The extract of the preceding paragraph, wherein the concentration of anyone D-amino acid comprises between 15 and 35% of the free amino acidconcentration.

The extract of any of the preceding paragraphs, wherein the extractcomprises between 6 and 75 mg/mL of one or more proteins.

The extract of the preceding paragraph, wherein the extract comprisesbetween 6 and 8 mg/mL of one or more proteins.

The extract of any of the preceding paragraphs, wherein the one or moreproteins have molecular weights of less than 30 kDa.

The extract of any of the preceding paragraphs, wherein the one or moreproteins have molecular weights of less than 10 kDa.

The extract of any of the preceding paragraphs, wherein the survivalrate of at least 8 mice with wild-type LPS sensitivity 13 days afterchallenge with Salmonella thyphimurium is at least 70%, wherein the doseof Salmonella thyphimurium is chosen such that the survival rate of atleast 8 control mice is 60% or lower.

The extract of the preceding paragraph, wherein the survival rate is atleast 80%.

The extract of the preceding paragraph, wherein the survival rate is atleast 90%.

The extract of any of the preceding paragraphs, wherein the extractcomprises less than 5000 ng of LPS equivalents according to a limulusamoebocyte lysate (LAL) chromogenic test.

A pharmaceutical composition comprising the extract of any of the aboveparagraphs.

A method of treating a subject suffering from or at risk of developing arespiratory disorder, comprising administering an effective amount ofany of the extracts of the above paragraphs to said subject.

The method of the preceding paragraph, wherein the subject is a human ordomestic mammal.

The method of either of the two preceding paragraphs, wherein therespiratory disorder or allergic condition is upper and lowerrespiratory infections, atopic dermatitis, nasopharyngitis, sinusitis,pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis,influenza, pneumonia, bronchopneumonia, bronchitis, lower respiratoryinfections, allergic rhinitis, allergic asthma, rhinitis,nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis,laryngotracheitis, bronchitis, obstructive pulmonary disease with acutelower respiratory infection, or obstructive pulmonary disease with acuteexacerbation.

A process for preparing an extract obtained from one or more bacterialspecies chosen from: Moraxella catarrhalis, Haemophilus influenzae,Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus sanguinis, StaphylococcusHemolyticus, Enterococcus faecalis, Streptococcus mutans, Streptococcusanginosus, Streptococcus mitis, Streptococcus salivarius, Neisseriasicca, Haemophilus parainfluenzae, Actinobacillus (Hemophilus)actinomycetemcomitans, and Eikenella corrodens, comprising:

(a) culturing each bacterial strain in a medium that does not pose arisk of prion diseases;(b) lysing each strain at an initial pH of greater than 12; and(c) passing the product of (b) at least once through a microfilter andat least once through an ultrafilter.

The process of the preceding paragraph, wherein the extract is obtainedfrom at least one strain of each of the following bacterial species:Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus sanguinis, Staphylococcus Hemolyticus, Enterococcusfaecalis, Streptococcus mutans, Streptococcus anginosus, Streptococcusmitis, Streptococcus salivarius, Neisseria sicca, Haemophilusparainfluenzae, Actinobacillus (Hemophilus) actinomycetemcomitans, andEikenella corrodens.

The process of the preceding paragraph, wherein the extract is obtainedfrom each of the following bacterial strains: Moraxella (Moraxella)catarrhalis 3622, Moraxella (Moraxella) catarrhalis 3625, Moraxella(Moraxella) catarrhalis I-045, Haemophilus influenzae 8467, Klebsiellapneumoniae ssp. ozaenae 5050, Klebsiella pneumoniae 204, Klebsiellapneumoniae 5056, Staphylococcus aureus I-049, Staphylococcus aureusI-050, Staphylococcus aureus I-051, Staphylococcus aureus I-052,Staphylococcus aureus I-053, Staphylococcus aureus I-054, Streptococcuspneumoniae 7465, Streptococcus pneumoniae 7466, Streptococcus pneumoniae7978, Streptococcus pneumoniae 10319, Streptococcus pyogenes 8191,Streptococcus sanguinis I-046, Streptococcus sanguinis I-047,Streptococcus sanguinis I-048, Staphylococcus Hemolyticus 11042,Enterococcus faecalis 103015, Streptococcus mutans 10449, Streptococcusanginosus 10713, Streptococcus mitis 12261, Streptococcus salivarius102503, Neisseria sicca 103345, Haemophilus parainfluenzae 7857,Actinobacillus (Hemophilus) actinomycetemcomitans 52.105, and Eikenellacorrodens 10596. The process of any of the preceding paragraphs, whereinthe lysis is carried out at an initial pH of greater than 12.5.

The process of any of the preceding paragraphs, wherein the lysis iscarried out at an initial pH of 12.6 to 13.4.

The process of any of the preceding paragraphs, wherein the lysis iscarried out for a period of from 40 hours to 10 days at a temperature of30-60° C.

The process of any of the preceding paragraphs, wherein the microfilteris 0.45 microns and the ultrafilter is 30 KDa.

The process of any of the preceding paragraphs, wherein part (c)comprises tangential flow filtration.

The process of the preceding paragraph, wherein the tangential flowfiltration is carried out for 5 to 15 cycles.

The process of any of the preceding paragraphs, further comprisingpassing the product of (c) through a second microfilter at 0.2 microns.

The process of any of the preceding paragraphs, wherein part (b) iscarried out with 10-120 g/l bacterial dry weight of material.

The process of any of the preceding paragraphs, wherein the tangentialflow filtration is performed as set forth in FIG. 1.

The process of any of the preceding paragraphs, wherein the tangentialflow filtration is performed as set forth in FIG. 1, in serpentine mode.

A product obtained by any of the processes of the preceding paragraphs.

A method of treating a subject suffering from or at risk of developing arespiratory disorder, comprising administering an effective amount ofany of the product of any one of the processes of the above paragraphsto said subject.

The method of the preceding paragraph, wherein the subject is a human ordomestic mammal.

1-48. (canceled)
 49. An extract from one or more bacterial specieschosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiellapneumoniae, Staphylococcus aureus, Streptococcus pneumoniae,Streptococcus pyogenes, and Streptococcus sanguinis, wherein saidextract comprises less than 100 μg/ml nucleic acids and at least 0.3mg/mL of saccharides.
 50. The extract of claim 49, wherein said extractcomprises 0.3-4.5 mg/mL, or 0.3-4 mg/mL, or 0.4-4 mg/mL, or 0.5-3.5mg/mL, or 0.6-3 mg/mL, or 0.3-1 mg/mL of saccharides.
 51. The extract ofclaim 49 wherein at least one saccharide is chosen amongmonosaccharides, disaccharides, polysaccharides, and lipopolysaccharides(LPS).
 52. The extract of claim 49, wherein said extract comprises 5-75mg/mL, or 10-65 mg/mL, or 20-45 mg/mL, or 20-40 mg/mL, or 5-20 mg/mL, or5-10 mg/mL, or 6-8 mg/mL of proteins, one or more of said proteinshaving a molecular weight of less than 30 kDa or less than 15 kDa, or 10kDa.
 53. The extract of claim 49, wherein one or more of the naturallyoccurring L-amino acids found in natural proteins of said extract areracemized into one or more D-amino acid.
 54. The extract of claim 53,wherein said one or more D-amino acid is chosen from aspartic acid,asparagine, glutamic acid, glutamine, serine, methionine, histidine,alanine, arginine, phenylalanine, tyrosine, leucine, lysine, valine, andthreonine.
 55. The extract of claim 53, wherein said extract comprisesamino acids, wherein said amino acids in the extract comprise between1-80%, or 10-45%, or 25-35% of D-amino acids.
 56. The extract of claim53, wherein said extract comprises 1.5 to 2.5 mg/mL, or 2 to 2.5 mg/mLor 1.5 to 2 mg/mL of amino acids in equivalents of glutamic acid (147.1g/mol).
 57. The extract of claim 49, wherein said one or more bacterialspecies are chosen among the following strains: Moraxella catarrhalis3622, Moraxella catarrhalis 3625, Moraxella catarrhalis I-045,Haemophilus influenzae 8467, Klebsiella pneumoniae ssp. ozaenae 5050,Klebsiella pneumoniae ssp. pneumoniae 204, Klebsiella pneumoniae ssp.pneumoniae 5056, Staphylococcus aureus I-049, Staphylococcus aureusI-050, Staphylococcus aureus I-051, Staphylococcus aureus I-052,Staphylococcus aureus I-053, Staphylococcus aureus I-054, Streptococcuspneumoniae 7465, Streptococcus pneumoniae 7466, Streptococcus pneumoniae7978, Streptococcus pneumoniae 10319, Streptococcus pyogenes 8191,Streptococcus sanguinis I-046, Streptococcus sanguinis I-047, andStreptococcus sanguinis I-048.
 58. The extract of claim 49, which doesnot pose any risk to prion diseases upon administration to a patient.59. A pharmaceutical composition comprising the extract of claim
 49. 60.A method of treating and/or preventing a respiratory disorder orallergic condition in a subject, comprising administering atherapeutically effective amount of the extract of claim 49 to saidsubject.
 61. The method of claim 60, wherein the subject is a human ordomestic mammal.
 62. The method of claim 60, wherein the respiratorydisorder or allergic condition is chosen from upper and lowerrespiratory infections, atopic dermatitis, nasopharyngitis, sinusitis,pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis,influenza, pneumonia, bronchopneumonia, bronchitis, lower respiratoryinfections, allergic rhinitis, allergic asthma, asthma, rhinitis,nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis,laryngotracheitis, bronchitis, obstructive pulmonary disease with acutelower respiratory infection, and obstructive pulmonary disease withacute exacerbation.
 63. A method of protecting a subject suffering froma respiratory disorder or allergic condition, or at risk of developing arespiratory disorder or allergic condition, comprising administering atherapeutically effective amount of the extract of claim 49 to saidsubject.
 64. The method of claim 63, wherein the respiratory disorder orallergic condition is chosen from upper and lower respiratoryinfections, atopic dermatitis, nasopharyngitis, sinusitis, pharyngitis,tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza,pneumonia, bronchopneumonia, bronchitis, lower respiratory infections,allergic rhinitis, allergic asthma, asthma, rhinitis, nasopharyngitis,pharyngitis, sinusitis, tonsillitis, laryngitis, laryngotracheitis,bronchitis, obstructive pulmonary disease with acute lower respiratoryinfection, and obstructive pulmonary disease with acute exacerbation.65. The extract of claim 49, obtainable by lysing one or more bacterialspecies chosen from Moraxella catarrhalis, Haemophilus influenzae,Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae,Streptococcus pyogenes, and Streptococcus sanguinis, at a pH of greaterthan
 12. 66. A process for preparing the extract of claim 49 from one ormore bacterial species chosen from Moraxella catarrhalis, Haemophilusinfluenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcuspneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis,comprising: (a) culturing each of said bacterial species in a mediumthat does not pose a risk of prion diseases; (b) lysing each species atan initial pH of greater than 12; and (c) passing the product of (b) atleast once through a microfilter and at least once through anultrafilter.
 67. The process of claim 66, wherein the lysis is carriedout for a period of 40 hours to 10 days at a temperature of 30 to 60° C.68. The process of claim 66, wherein the lysis is carried out for aperiod of 20 to 240 hours at a temperature 30 to 40° C.